Human enzymes of the metalloprotease family

ABSTRACT

This invention relates to newly identified polypeptides which have zinc metalloprotease activities and are referred to as IGS5, and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be stimulators and/or inhibitors which are useful in therapy, and to production of such polypeptides and polynucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of application Ser. No.10/870,003, filed Jun. 18, 2004, now U.S. Pat. No. 7,083,965, which inturn was a division of application Ser. No. 10/147,928, filed May 20,2002, now U.S. Pat. No. 6,855,532, which was a continuation ofinternational patent application no. PCT/EP00/11532, filed Nov. 17,2000, designating the United States of America, the entire disclosure ofwhich is incorporated herein by reference. Priority is claimed based onEuropean patent application no. 99 20 3862.0, filed Nov. 19, 1999; Dutchpatent application no. 1013616, filed Nov. 19, 1999; Europeanapplication no. 00201937.0, filed May 31, 2001; and Dutch patentapplication no. 1015356, filed May 31, 2000.

BACKGROUND OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be stimulators and/or inhibitorswhich are potentially useful in therapy, and to production of suchpolypeptides and polynucleotides. More particularly, the polypeptidesand polynucleotides of the present invention relate to enzymes which aremembers of the metalloprotease family of polypeptides or of otherstructurally and functionally related polypeptides. These enzymes arehereinafter referred to as IGS5.

The invention also relates to inhibiting or stimulating/activating theaction of such polypeptides and polynucleotides, to a vector containingsaid polynucleotides and to a host cell containing such vector. Theinvention further relates to a method for screening compounds capable ofstimulating or inhibiting said IGS5 enzymes.

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces “functional genomics,” that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on “positional cloning.” Aphenotype, such as a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencingtechnologies and various tools of bioinformatics to identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterize further genes and their related polypeptides/proteins, astargets for drug discovery.

Among the polypeptides of interest in drug discovery there aremetalloproteases and other structurally and functionally relatedenzymes. Several diseases have been identified where metalloproteasesplay a critical role in the pathology of the disease. For example, anumber of zinc metalloproteases or other structurally and functionallyrelated enzymes have been identified and characterized in the state ofthe art, and it has become apparent that the participation of theseenzymes, e.g. zinc metalloproteases, plays a role in a diverse array ofbiological functions encompassing both normal and disease situations.Zinc metalloproteases are subset of such enzymes whose catalyticfunctions are critically dependent on the zinc ion at the active site.This group of enzymes, which comprises various families classified onthe basis of both sequence and structural information, are for exampledescribed to be intimately involved in such processes as embryonicdevelopment, cartilage and bone formation, processing of peptidehormones, reproduction, cardiovascular diseases, arthritis and cancer.Already active site-directed inhibitors of some of the zincmetalloproteases are being used therapeutically as e.g.antihypertensives.

On the basis of sequence and structural information around the zincbinding site of the zinc metalloproteases these enzymes may beclassified into several families which may be further classified intosuperfamilies such as the “metzincins” (astacin, serratia, reprolysin,matrixin), the “gluzincins” (thermolysin, neprilysin, angiotensinconverting enzyme, aminopeptidase), or the “zincins” comprising thesuperfamilies of metzincins and gluzincins. Such grouping not only aidsin the elucidation of common catalytic and biosynthetic processingmechanisms, but also is invaluable in elucidating the function(s) ofnewly identified proteins which possess similar zinc binding motifs.Some individual examples of metalloproteases, e.g. zinc enzymes, alreadyidentified in the state of the art comprise neprilysin, endothelinconverting enzyme, angiotensin converting enzyme, thermolysin,aminopeptidase, astacin, serratia, reprolysin, matrixin, insulinase,carboxypeptidase and DD-carboxypeptidase.

From the above evidence based on the state of the art it is apparentthat metalloproteases and other structurally and functionally relatedenzymes play key roles in health and disease. Thus there is a continuedneed to further uncover important functions and potential therapeuticapplications for this group of enzymes and to provide novelmetalloproteases with the subsequent development of novel syntheticstimulators (activators) or inhibitors, which can help provide newtreatments for a variety of diseases of socio-economic importance.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to IGS5, in particular toIGS5 polypeptides and IGS5 polynucleotides, preferably those related tothe human species, to recombinant materials and methods for theirproduction.

In another aspect, the invention relates to methods for using suchpolypeptides, polynucleotides and recombinant materials, including thetreatment of diseases in which metalloproteases or structurally andfunctionally related enzymes play a critical role in the pathology.

Examples of diseases, in context of which the use of the polypeptidesand polynucleotides of the present invention is thought to be useful,include, but are not limited to: CNS disorders, including schizophrenia,episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsivedisorder (OCD), post traumatic stress disorder (PTSD), phobia and panic,major depressive disorder, bipolar disorder, Parkinson's disease,general anxiety disorder, autism, delirium, multiple sclerosis,Alzheimer disease/dementia and other neurodegenerative diseases, severemental retardation and dyskinesias, such as Huntington's disease orGilles dela Tourett's syndrome, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovascular diseasesincluding heart failure, angina pectoris, arrhythmias, myocardialinfarction, cardiac hypertrophy, hypotension, hypertension—e.g.essential hypertension, renal hypertension, or pulmonary hypertension,thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoidhemorrhage, cerebral ischemia, cerebral infarction, peripheral vasculardisease, Raynaud's disease, kidney disease—e.g. renal failure;dyslipidemias; obesity; emesis; gastrointestinal disorders includingirritable bowel syndrome (IBS), inflammatory bowel disease (IBD),gastroesophagal reflux disease (GERD), motility disorders and conditionsof delayed gastric emptying, such as postoperative or diabeticgastroparesis, and diabetes, ulcers—e.g. gastric ulcer; diarrhoea; otherdiseases including osteoporosis; inflammations; infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; chemotherapy inducedinjury; tumor invasion; immune disorders; urinary retention; asthma;allergies; arthritis; benign prostatic hypertrophy; endotoxin shock;sepsis; complication of diabetes mellitus.

In a further aspect, the invention relates to methods for identifyingagonists and antagonists or inhibitors using the materials provided bythe invention, and treating conditions associated with IGS5 imbalancewith the identified compounds.

In a still further aspect, the invention relates to diagnostic assaysfor detecting diseases associated with inappropriate IGS5 activity orlevels.

The Polypeptides of the present invention are in particular of interestin the context of cardiovascular diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of the relative positions of thedifferent cDNA clones that were isolated and fully sequenced to generatethe partial IGS5 consensus cDNA sequence. PCR primers that were used for5′ RACE and semi-homology PCR cloning are indicated and have beendescribed in this document (indicated by the respective IP#). IGS5CONSdenotes the consensus contig that was obtained after merging allobtained sequences. The 691 amino acids long open reading frame presentin the IGS5 contig, that is postulated to contain the ectodomain of theIGS5 enzyme (IGS5DNA, IGS5PROT) is indicated with open boxes(“.quadrature..quadrature.”). The part of the aligned EST sequences(accession no AA524283, AI088893, AI217369 and AI380811) that bearshomology to members of the NEP/ECE family is indicated with “+==+”(IGS5EST). “bp”=base pairs.

FIG. 2 Schematic representation of the relative positions of thedifferent cDNA clones that were isolated and fully sequenced to generatethe IGS5DNA1 and IGS5DNA2 cDNA sequences. PCR primers that were used forPCR, 5′ RACE and semi-homology PCR cloning are indicated and have beendescribed in this document (indicated by the respective IP#). IGS5CONS1and IGS5CONS2 denote the 2 different consensus contigs that wereobtained after merging all obtained sequences. IGS5DNA1 and IGS5DNA2denote the open reading frames present in IGS5CONS 1 and IGS5CONS2respectively (“**”). The part of the aligned EST sequences (accessionno. AA524283, AI088893, AI217369 and AI 380811) that bears homology tomembers of the NEP/ECE family is indicated with “+==+” (IGS5EST).“bp”=base pairs. The 78 bp fragment identified within genomic cloneIGS5/S1 is denoted as “IGS5S1/78 bp.” The absence of the 78 bp alternateexon sequence within clones YCE231, YCE233 and YCE235 and withinIGS5CONS2 and IGS5DNA2 is indicated by a gap.

FIG. 3A and 3B Master Blot™ analysis of the IGS5 gene.

FIG. 4 Sequence of the 180 bp fragment, encoding the POMC signalsequence, the Gly-Ser linker, the His6 tag and the start of the IGS5ectodomain sequence, assembled by overlap PCR using differentoligonucleotides. (*silent mutation (bp 57 of the pomc signalsequence)).

FIG. 5 Plasmid map of vector pAcSG2SOLhuIGS5His6.

FIG. 6 Predicted protein sequence of the mature recombinant solubleHis-tagged human IGS5, as expressed in Sf9 cells upon infection withrecombinant baculovirus IGBV73 (after cleavage of the 26AA long POMCsignal sequence). Potential N-glycosylation sites are underlined.

FIG. 7 Deglycosylation study—Western blot analysis. 72 h CM harvest ofthe infection with the 3 recombinant soluble His6IGS5 clones (clone 1:lanes 1 to 3, clone2: lanes 4 to 6, clone 3: lanes 7 to 9) was treatedas described with and without addition of N-glycosidase F. 10 μl CMequivalent was loaded on gel versus 20 μl of the non-treated CM as acontrol. Detection was performed with anti-His antibody (21E1B4EPR300,Innogenetics, 1 μg/ml final concentration). Second antibody was rabbitanti mouse-Alkaline Phosphatase conjugated (Sigma A-1902). Revelation ofthe bands was done with NBT-BCIP. Mr marker is the Biolabs broad rangeMW marker (cat. no. 7707S).

FIG. 8 SDS PAGE analysis under reducing conditions (+DTT) on 12.5%PHASTgel (Pharmacia; 4 μl/slot) of the Lentil chromatography steps.Proteins were visualised by silver staining.

FIG. 9 Western blot analysis of IGS5 at different stages of thepurification procedure. Samples were separated on a 7.5% Minigel (BioradMINI-Protean II) and analyzed via Western blot using the anti His6primary mab 21E1B4, followed by an alkaline phosphatase conjugatedrabbit anti mouse Ig as a secondary antibody and detection by nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). The columnunder DTT denotes whether the proteins were reduced or not with1,4-dithio-DL-threitol (DTT).

FIG. 10 SDS PAGE analysis under reducing conditions (+DTT) on 12.5%PHASTgel (Pharmacia, 4 μl slots) of different imidazole elution pools ofthe Zn-IMAC chromatography of pool 1 from the lentil chromatographyeluate. Proteins were visualised by silver staining.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“IGS5” refers, among others, to a polypeptide comprising the amino acidsequence set forth in one of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6,or respective variants thereof. Thus “IGS5” particularly includesIGS5PROT, IGS5PROT1 and IGS5PROT2 (see below).

“Enzyme Activity” or “Biological Activity” refers to the metabolic orphysiologic function of said IGS5 including similar activities orimproved activities or these activities with decreased undesirable sideeffects. Also included are antigenic and immunogenic activities of saidIGS5.

“IGS5-gene” refers to a polynucleotide comprising the nucleotidesequence set forth in one of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5,or respective variants, e.g. allelic variants, thereof and/or theircomplements.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of a Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural stateand/or separated from the natural environment. Thus, if an “isolated”composition or substance that occurs in nature has been “isolated,” ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated,” as the term is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol; cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formulation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,“Proteins—Structure and Molecular Properties,” 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., “Post-translationalProtein Modifications: Perspectives and Prospects,” pp. 1–12 in“Post-translational Covalent Modification of Proteins,” B. C. Johnson,Ed., Academic Press, New York, 1983; Seifter et al., “Analysis forprotein modifications and nonprotein cofactors,” Meth. Enzymol. (1990)182:626–646; and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging,” Ann. NY Acad. Sci. (1992) 663:48–62).

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide respectively, but retainsessential properties. A typical variant of a polynucleotide differs innucleotide sequence from another, reference polynucleotide. Changes inthe nucleotide sequence of the variant may or may not alter the aminoacid sequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,and deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity,” as known as a measure of identity in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences, e.g. ingenerally by alignment of the sequences so that the highest order matchis obtained. Thus “Identity” and or the alternative wording “Similarity”has an art-recognized meaning and can be readily calculated by knownmethods, including but not limited to those described in “ComputationalMolecular Biology,” Lesk, A. M., Ed., Oxford University Press, New York,1988; “Biocomputing: Informatics and Genome Projects,” Smith, D. W.,Ed., Academic Press, New York, 1993; “Computer Analysis of SequenceData,” Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press,New Jersey, 1994; “Sequence Analysis in Molecular Biology,” von Heinje,G., Academic Press, 1987; “Sequence Analysis Primer,” Gribskov, M. andDevereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403–410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403–410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity. A publicly available program useful to determineidentity or similarity of polypeptide sequences or polynucleotidesequence, respectively, is known as the “gap” program from GeneticsComputer Group, Madison Wis., which is usually run with the defaultparameters for comparisons (along with no penalty for end gaps). Thepreferred (i.e. default) parameters for polypeptide sequence comparisoninclude the following: Algorithm as described by Needleman and Wunsch,J. Mol. Biol. 48: 443–453 (1970); Comparison Matrix BLOSSUM62 fromHentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915–10919(1992); Gap Penalty: 12; Gap Length Penalty: 14. The preferred (i.e.default) parameters for polynucleotide sequence comparison include thefollowing: Algorithm as described by Needleman and Wunsch, J. Mol. Biol.48: 443–453 (1970); Comparison Matrix: matches=+10, mismatch=0; GapPenalty: 50; Gap Length Penalty: 3. The word “homology” may substitutefor the word “identity.”

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence, for example to a reference nucleotid sequence selected fromthe group of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, is intended thatthe nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the respectivereference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence,or in a number of nucleotides of up to 5% of the total nucleotides inthe reference sequence there may be a combination of deletion, insertionand substitution. These mutations of the reference sequence may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example 95% “identity” to a reference amino acid sequence,for example to a reference amino acid sequence selected from the groupof SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, is intended that the aminoacid sequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the respective reference aminoacid. In other words, to obtain a polypeptide having an amino acidsequence at least 95% identical to a reference amino acid sequence, upto 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino- or carboxy-terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a subject sequence. Such relatedness may be quantified by determiningthe degree of identity and/or similarity between the sequences beingcompared as herein described. Falling within this generic term are theterms “ortholog,” meaning a polynucleotide or polypeptide that is thefunctional equivalent of a polynucleotide or polypeptide in anotherspecies, and “paralog” meaning a functionally similar sequence whenconsidered within the same species. Hence, in humans for example, withinthe family of endothelin converting enzymes ECE-1 is a paralog of theother members, e.g. of ECE-2.

“Fusion protein” refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. This term may be illustrated forexample by fusion proteins comprising various portions of constantregion of immunoglobulin molecules together with another human proteinor part thereof. In many cases, employing an immunoglobulin Fc region asa part of a fusion protein is advantageous for use in therapy anddiagnosis resulting in, for example, improved pharmacokinetic properties(see, e.g., EP-A 0232 262). On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

Polypeptides of the Invention

The present invention relates to IGS5 polypeptides (or IGS5 enzymes,e.g. to IGS5PROT, IGS5PROT1 or IGS5PROT2, respectively), in particularto human IGS5 polypeptides (or human IGS5 enzymes), and also to IGS5polypeptide fragments comprising a substantial portion of said entireIGS5 polypeptide. Thus, in a first aspect, the IGS5 polypeptides of thepresent invention include isolated polypeptides, in particular isolatedhuman species polypeptides, comprising an amino acid sequence which hasat least 70% identity, preferably at least 80% and in particular atleast 85% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, still more preferably at least 97–99%identity, to one of that selected from the group of SEQ ID NO:2, SEQ IDNO:4 SEQ and SEQ ID NO:6, over the entire length of the respective SEQID NO:2, SEQ ID NO:4 SEQ and SEQ ID NO:6. Such polypeptides includethose comprising one of the amino acid sequences selected from the groupof SEQ ID NO:2, SEQ ID NO:4 SEQ and ID NO:6.

In a second aspect, the IGS5 polypeptides of the present inventioninclude isolated polypeptides, in particular isolated human IGS5polypeptides, having an amino acid sequence of at least 70% identity,preferably at least 80% and in particular at least 85% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, still more preferably at least 97–99% identity, to one of theamino acid sequences selected from the group of SEQ ID NO:2, SEQ ID NO:4SEQ and ID NO:6, over the entire length of the respective SEQ ID NO:2,SEQ ID NO:4 SEQ and ID NO:6. Such polypeptides include the IGS5polypeptide of SEQ ID NO:2, of SEQ ID NO:4 and SEQ ID NO:6,respectively.

Further polypeptides of the present invention include isolated IGS5polypeptides comprising the sequence contained in one of SEQ ID NO:2,SEQ ID NO:4 SEQ and ID NO:6, and which in particular are human speciespolypeptides. Polypeptides of the present invention are members of themetalloprotease family of polypeptides. They are of interest becauseseveral dysfunctions, disorders or diseases have been identified wheremetalloproteases play a critical role in the pathology of the disease.Examples of the diseases, in context of which the polypeptides andpolynucleotides of the present invention are thought to be useful,include amongst others: CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation and dyskinesias, such as Huntington's disease or Gilles delaTourett's syndrome, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovascular diseasesincluding heart failure, angina pectoris, arrhythmias, myocardialinfarction, cardiac hypertrophy, hypotension, hypertension—e.g.essential hypertension, renal hypertension, or pulmonary hypertension,thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoidhemorrhage, cerebral ischemia, cerebral infarction, peripheral vasculardisease, Raynaud's disease, kidney disease—e.g. renal failure;dyslipidemias; obesity; emesis; gastrointestinal disorders includingirritable bowel syndrome (IBS), inflammatory bowel disease (IBD),gastroesophagal reflux disease (GERD), motility disorders and conditionsof delayed gastric emptying, such as post-operative or diabeticgastroparesis, and diabetes, ulcers—e.g. gastric ulcer; diarrhoea; otherdiseases including osteoporosis; inflammations; infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; chemotherapy inducedinjury; tumor invasion; immune disorders; urinary retention; asthma;allergies; arthritis; benign prostatic hypertrophy; endotoxin shock;sepsis; complication of diabetes mellitus. The Polypeptides of thepresent invention are in particular of interest in the context ofcardiovascular diseases.

Furthermore, the IGS5 polypeptides of the invention are also of interestfor identifying stimulators or inhibitors of these polypeptides, forproviding diagnostic assays for detecting diseases associated withinappropriate IGS5 activity or levels, and for treating conditionsassociated with IGS5 imbalance with compounds identified to bestimulators or inhibitors. Hence, the IGS5 polypeptides of the inventionmay be used for designing or screening for selective stimulators orinhibitors, and thus can lead to the development of new drugs. Theproperties of the IGS5 polypeptides, in particular of the human speciesIGS5 polypeptides, of the present invention are hereinafter referred toas “IGS5 activity” or “IGS5 polypeptide activity” or “biologicalactivity of IGS5.” Also included amongst these activities are antigenicand immunogenic activities of said IGS5 polypeptides, in particular theantigenic and immunogenic activities of one of the polypeptides selectedfrom the group of SEQ ID NO:2, SEQ ID NO:4 SEQ and ID NO:6. Preferably,a polypeptide of the present invention exhibits at least one biologicalactivity of IGS5, preferably of human IGS5.

The IGS5 polypeptides of the present invention may be in the form of a“mature” protein or may be a part of a larger protein such as aprecursor or a fusion protein. It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr, among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5–10, 1–5, 1–3, 1–2 or 1 amino acids aresubstituted, deleted, or added in any combination.

The present invention furthermore pertains to fragments of the IGS5polypeptides, in particular to IGS5 polypeptide fragments comprising asubstantial portion of the entire IGS5 polypeptide. A fragment is apolypeptide having an amino acid sequence that entirely is the same aspart, but not all, of the amino acid sequence of the aforementioned IGS5polypeptides. As with IGS5 polypeptides, fragments may be“free-standing,” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region.Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of IGS5 polypeptides, except for deletion of acontinuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Other preferred fragments are biologicallyactive fragments. Biologically active fragments are these that mediateenzyme activity, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Also included arethose that are antigenic or immunogenic in an animal, especially in ahuman.

With regard to the variant of the invention pertaining to polypeptidefragments comprising a substantial portion of the entire IGS5polypeptide as shown in one of SEQ ID NO:2, SEQ ID NO:4 SEQ and ID NO:6,the term “substantial” has the meaning that the fragment of the IGS5polypeptide has in particular a size of at least about 50 amino acids,preferably a size of at least about 100 amino acids, more preferably asize of at least about 200 amino acids, most preferably a size of atleast about 300 amino acids. In this context “about” includes theparticularly recited sizes larger or smaller by several, 5, 4, 3, 2 or 1amino acids. The IGS5 polypeptide fragments according to the inventionpreferably show at least to some extent at least one of the propertieswhich are characteristic for the IGS5 polypeptides themselves.

With regard to the IGS5 polypeptides of the present invention it wasfound that they may be involved in the metabolism of biologically activepeptides. In particular it was found that these IGS5 polypeptides aremetalloprotease type enzymes which may act on a variety of vasoactivepeptides. Vasoactive peptides known in the state of the art includeatrial natriuretic peptide (ANP), bradykinin, big endothelin (Big ET-1),endothelin (ET-1), substance P, and angiotensin-1 In the context of thepresent invention it was found that the IGS5 ectodomain, which is anovel human metalloprotease, hydrolyzes e.g. in vitro a variety of saidvasoactive peptides including Big ET-1, ET-1, ANP and bradykinin.

Furthermore, the IGS5 metalloprotease type enzymes of the presentinvention may be inhibited by reference compounds that are used todetermine the inhibition properties with regard to enzymes havingECE/NEP-characteristics, e.g. inhibition by compounds such asphosphoramidon. No inhibition of IGS5 is observed for referencecompounds that specifically inhibit NEP, e.g. no inhibition of IGS5 bycompounds such as thiorphan. Nor any inhibition of IGS5 is observed forreference compounds that specifically inhibit ECE, e.g. no inhibition ofIGS5 by compounds such as the selective ECE inhibitor CGS-35066 (DeLombart et al., J. Med. Chem. 2000, Feb. 10; 43(3):488–504). Theinhibition data of these reference compounds with regard to theinhibition of the IGS5 metalloprotease type enzymes of the presentinvention are further described in the experimental part below, inparticular in Example 7.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

In another aspect, the present invention relates to IGS5 polynucleotides(e.g. to IGS5DNA, IGS5DNA1 or IGS5DNA2, respectively), in particular tohuman IGS5 polynucleotides. Such polynucleotides include isolatedpolynucleotides, preferably isolated human species polynucleotides,comprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% and in particular at least85% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, to one of the amino acid sequences selected fromthe group of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, over the entirelength of the respective SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Inthis regard, polynucleotides encoding polypeptides which have at least97% identity are highly preferred, whilst those with at least 98–99%identity are more highly preferred, and those with at least 99%, inparticular 99.9%, identity are most highly preferred. Suchpolynucleotides include polynucleotides comprising the nucleotidesequence contained in one of the SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5, encoding the respective polypeptide of SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6.

In a variant of this aspect, the polynucleotides of the presentinvention include isolated polynucleotides, in particular isolated humanpolynucleotides, comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% and in particular at least 85%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, to a nucleotide sequence encoding one of thepolypeptides selected from the group of SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:6, over the entire coding region. In this regard, polynucleotideswhich have at least 97% identity are highly preferred, whilst those withat least 98–99% identity are more highly preferred, and those with atleast 99%, in particular 99.9%, identity are most highly preferred.

Further polynucleotides of the present invention include isolatedpolynucleotides, in particular isolated human polynucleotides,comprising a nucleotide sequence which has at least 70% identity,preferably at least 80% and in particular at least 85% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, to one of the nucleotide sequences selected from the group ofSEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, over the entire length of therespective SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. Particularly,polynucleotides of the present invention include isolatedpolynucleotides having a nucleotide sequence of at least 70% identity,preferably at least 80% and in particular at least 85% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, to the respective reference nucleotide sequence over theentire length of the reference nucleotide sequence. In this regard,polynucleotides which comprise or have a nucleotide sequence of at least97% identity to one of the nucleotide sequences selected from the groupof SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 are highly preferred, whilstthose with at least 98–99% identity, are more highly preferred, andthose with at least 99%, in particular 99.9%, identity are most highlypreferred. Such polynucleotides include a polynucleotides comprising oneof the polynucleotides of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, aswell as the polynucleotides of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5themselves, which in particular are human species polynucleotides. Theinvention also provides polynucleotides which are complementary to allthe above described polynucleotides.

The nucleotide sequence of SEQ ID NO:1 (designated “IGS5DNA”) is a cDNAsequence from human origin (Homo sapiens) with a length of 2076nucleotides and comprises a polypeptide encoding sequence (fromnucleotide no. 1 to no. 2073) encoding a polypeptide of 691 amino acids,the polypeptide of SEQ ID NO:2 (designated “IGS5PROT”). The nucleotidesequence encoding the polypeptide of SEQ ID NO:2 may be identical to thepolypeptide encoding sequence contained in SEQ ID NO:1 or it may be asequence other than the one contained in SEQ ID NO:1, which, as a resultof the redundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2.

The nucleotide sequence of SEQ ID NO:3 (designated “IGS5DNA1”) is a cDNAsequence from human origin (Homo sapiens) with a length of 2340nucleotides (including the stop codon tag) and comprises a polypeptideencoding sequence (from nucleotide no. 1 to no. 2337) encoding apolypeptide of 779 amino acids, the polypeptide of SEQ ID NO:4(designated “IGS5PROT1”). The nucleotide sequence encoding thepolypeptide of SEQ ID NO:4 may be identical to the polypeptide encodingsequence contained in SEQ ID NO:3 or it may be a sequence other than theone contained in SEQ ID NO:3, which, as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:4.

The nucleotide sequence of SEQ ID NO:5 (designated “IGS5DNA2”) is a cDNAsequence from human origin (Homo sapiens) with a length of 2262nucleotides (including the stop codon tag) and comprises a polypeptideencoding sequence (from nucleotide no. 1 to no. 2259) encoding apolypeptide of 753 amino acids, the polypeptide of SEQ ID NO:6(designated “IGS5PROT2”). The nucleotide sequence encoding thepolypeptide of SEQ ID NO:6 may be identical to the polypeptide encodingsequence contained in SEQ ID NO:5 or it may be a sequence other than theone contained in SEQ ID NO:5, which, as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:6.

The characteristics of the type of polypeptides encoded by thepolynucleotides of the invention are described in more detail below.

Biological and Pharmacological Features of Metalloproteases

The polypeptides of the present invention, and in particular those beinghuman species polypeptides, are structurally and functionally related toother proteins of the metalloprotease family, e.g. showing homologyand/or structural similarity with metalloproteases or related enzymes,such as e.g. matrix metalloproteases (MMPs), angiotensis convertingenzyme (ACE), endothelin converting enzyme (ECE) or neutralendopeptidase (NEP), respectively. Thus, for example, the polypeptide ofthe SEQ ID NO:2 is structurally and functionally related to otherproteins of the metalloprotease family, having homology and/orstructural similarity with enzymes such as NEP or ECE (e.g. ECE-1), andin particular with NEP. Thus, preferred polypeptides and polynucleotidesof the present invention are expected to have, inter alia, similarbiological functions/properties to their homologous polypeptides andpolynucleotides. Furthermore, preferred polypeptides and polynucleotidesof the present invention have at least one IGS5 activity.

The general features of metalloproteases and their activities, inparticular with regard to the present invention, are already describedabove. For further understanding of the nature and characteristics ofthe polypeptides and polynucleotides of the present invention, inparticular the function of these polypeptides and polynucleotides, somemore specific features of each of the enzymes like MMPs, ACE, ECE orNEP, respectively, are summarized as follows.

Matrix metalloproteases (MMPs), also designated matrixins, are a familyof zinc metalloproteases that function in the turnover of components ofthe extracellular matrix. To date, several members of the matrixinfamily have been identified in humans. MMPs are synthesized and secretedfrom a number of cell types such as fibroblasts, epithelial cells,phagocytes, lymphocytes and cancer cells. MMPs are synthesized aspre-pro-enzymes which are destined to be secreted as proenzymes from allproducing cells except neutrophils. Under physiological conditions theseenzymes play central roles in morphogenesis, tissue remodelling andresorption. In excess, they participate in the destruction of theextracellular matrix associated with many connective tissue diseasessuch as in arthritis, periodontitis, glomerulonephritis, and with cancercell invasion and metastasis. Thus, the MMPs play a central role, forexample, in the normal embryo-genesis and tissue remodelling and in manydiseases such as arthritis, cancer, periodontitis, glomerulonephritis,encephalomyelitis, atherosclerosis and tissue ulceration. The importanceof the matrixins in both physiological and pathological catabolism ofconnective tissue matrix has been emphasized, because little MMPactivity can be detected in normal steady-state tissues, but thesynthesis of many MMPs is transcriptionally regulated by inflammatorycytokines, hormones, growth factors and on cellular transformation. Thebiological activities of MMPs are further controlled extracellularlyduring steps in their activation from inactive precursors (proMMPs), aswell as through interaction with the extracellular substratum andendogenous inhibitors. The MMPs are an important class of zinc-dependentmetalloproteases involved in degradation and remodeling of theextracellular matrix. Inhibitors of these enzymes have therapeuticpotential in e.g. cancer, arthritis, osteoporosis and Alzheimer'sdisease, and several of these inhibitors are under clinical evaluation.

Angiotensin I Converting Enzyme (ACE; peptidyl dipeptidase A; EC3.4.15.1) is a member of the angiotensin converting enzyme family ofzinc metalloproteases. ACE is primarily expressed at the surface ofendothelial, epithelial and neuroepithelial cells (somatic ACE) as anectoenzyme, meaning that it is anchored to the plasma membrane with thebulk of its mass, including its catalytic site/s, facing theextracellular milieu. ACE is found in the plasma membrane of vascularendothelial cells, with high levels found at the vascular endothelialsurface of the lung such that the active sites of ACE are posed tometabolize circulating substrates. In addition to the endotheliallocation of ACE, the enzyme is also expressed in the brush borders ofabsorptive epithelia of the small intestine and the kidney proximalconvoluted tubule. ACE is also found in mononuclear cells, such asmonocytes after macrophage differentiation and T-lymphocytes, and infibroblasts. In vitro autoradiography, employing radiolabelled specificACE inhibitors, and immunohistochemical studies have mapped theprincipal locations of ACE in the brain. ACE was found primarily in thechoroid plexus, which may be the source of ACE in cerebrospinal fluid,ependyma, subfornical organ, basal ganglia (caudate-putamen and globuspallidus), substantia nigra and pituitary. A soluble form of ACE hasbeen detected in many biological fluids such as serum, seminal fluid,amniotic fluid and cerebrospinal fluid. The soluble form of ACE appearsto be derived from the membrane-bound form of the enzyme in endothelialcells. A main physiological activity of ACE is that it cleaves theC-terminal dipeptide from angiotensin I to produce the potentvasopressor peptide angiotensin II and inactivates the vasodilatorypeptide bradykinin by the sequential removal of two C-terminaldipeptides. As a consequence of the involvement of ACE in the metabolismof these two vasoactive peptides angiotensin II and bradykinin, ACE hasbecome a crucial molecular target in the treatment of hypertension andcongestive heart failure. This has led to the development of highlypotent and specific ACE inhibitors which have become clinicallyimportant and widespread as orally active drugs to control theseconditions of hypertension and congestive heart failure. Whilst themetabolism of vasoactive peptides remains the best known physiologicalfunction of ACE, the enzyme has been also implicated in a range of otherphysiological processes unrelated to blood pressure regulation such asimmunity, reproduction and neuropeptide metabolism due to thelocalization of ACE and/or the in vitro cleavage of a range ofbiologically active peptides.

Neutral Endopeptidase (NEP, neprilysin, EC 3.4.24.11) is a zincmetalloprotease and classified as a member of the neprilysin family. NEPwas first isolated from the brush border membranes of rabbit kidney.Later, an NEP-like enzyme was identified in rat brain as being involvedin the degradation of the opioid peptides, enkephalins. The cloning ofthe ectoenzyme NEP and subsequent site-directed mutagenesis experimentshave shown that, as well as having a similar specificity to thermolysin,it also has a similar active site organization. NEP also shows athermolysin-like specificity for cleaving peptides on the N-terminalside of hydrophobic residues. With regard to the general distribution ofNEP it has been determined in the brain and spinal cord, and lesion andelectron microscopic studies generally support a predominantly neuronallocalization of NEP, although the enzyme could be present onoligodendrocytes surrounding the fibers of the striato-pallidal andstriato-nigral pathways and on Schwann cells in the peripheral nervoussystem. NEP does not appear to be concentrated on specific membraneinterfaces such as the synapse, but is rather uniformly distributed onthe surface of neuronal perikarya and dendrites. In the periphery, NEPis particularly abundant in the brush border membranes of the kidney andintestine, the lymph nodes and the placenta, and is found in lowerconcentrations in many other tissues including the vascular wall of theaorta. By finding that the common acute lymphoblastic leukemia antigenwas NEP, it was also shown in the state of the art that the enzyme istransiently present at the surface of lymphohaematopoietic cells andelevated levels are found on mature lymphocytes in certain diseasestates. The clinical interest in NEP, in particular the interest in NEPinhibitors as potential clinical agents derives from the actions of NEP,in conjunction with another zinc metalloprotease, the aminopeptidase N(APN, membrane alanyl aminopeptidase, EC 3.4.11.2), in degrading theenkephalins and also from its role in degrading atrial natriureticpeptide (ANP). For example, it is known that dual inhibitors of NEP andangiotensin converting enzyme (ACE) are potent antihypertensives,resulting from simultaneously increasing the circulating levels ofatrial natriuretic peptide, due to NEP inhibition, and decreasing thecirculating levels of angiotensin II, due to ACE inhibition. Furtherinterest in the clinical potential of NEP inhibitors came when theperipheral enzyme was shown to degrade the circulating natriuretic anddiuretic peptide, atrial natriuretic peptide. NEP inhibitors weretherefore investigated for their antihypertensive properties. From afurther example it is known that inhibition of enkephalin metabolism bythe synthetic NEP inhibitor, thiorphan, gave naloxone-reversibleantinociceptive responses in mice. This opened the possibility that, byincreasing the levels of endogenous opioids in the regions of theirtarget receptors, an analgesia could be obtained relatively free of theside-effects of morphine or other classical opiate drugs. It wasrealized that in order to achieve any significant effect, otherenkephalin-metabolizing enzymes also had to be inhibited, in particularthe aminopeptidase N (APN). Such dual NEP/APN inhibitors completelyblock enkephalin metabolism and have strong antinociceptive properties.

Endothelin Converting Enzyme (ECE) catalyses the final step in thebiosynthesis of the potent vasoconstrictor peptide endothelin (ET). Thisinvolves cleavage of the Trp-Val bond in the inactive intermediate, bigendothelin. ECE-1 is a zinc metalloprotease which is homologous withneutral endopeptidase (NEP; neprilysin; EC 3.4.24.11, see above). LikeNEP, ECE-1 is inhibited by the compound phosphoramidon and is a type IIintegral membrane protein. Unlike NEP, however, ECE-1 exists as adisulfide-linked dimer and is not inhibited by other NEP inhibitors suchas thiorphan. Immunocytochemical studies indicate a predominantcell-surface location for ECE-1 where it exists as an ectoenzyme. ECE-1is localized to endothelial cells and some secretory cells, e.g..beta.-cells in the pancreas, and in smooth muscle cells. Potent andselective inhibitors of ECE, or dual inhibitors of ECE and NEP, may havetherapeutic applications in cardiovascular and renal medicine.Endothelin (ET) which is a 21 amino acid bicyclic peptide containing twointramolecular disulfide bonds, is one of the most potentvasoconstricting peptides identified to date and administration toanimals results in a sustained increase in blood pressure emphasizingits potential role in cardiovascular regulation. The endogenousproduction of ET-1 in humans contributes to the maintenance of basalvascular tone. The endothelin system and related enzymes like ECEtherefore represent a likely candidate for the development of novelpharmaceutical agents. Thus, the clinical interest in ECE, in particularthe interest in ECE inhibitors as potential clinical agents derives fromthe actions of ECE, in particular in the context of the biosynthesis ofET. Consequently, compounds showing a significant endothelin convertingenzyme inhibitory activity are useful in treating and preventing variousdiseases which are induced or suspected to be induced by ET, such as forexample, cardiovascular diseases including heart failure, anginapectoris, arrhythmias, myocardial infarction, cardiac hypertrophy,hypotension, hypertension—e.g. essential hypertension, renalhypertension, or pulmonary hypertension, thrombosis, arteriosclerosis,cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; asthma; stroke, Alzheimer's disease;complication of diabetes mellitus; ulcer such as gastric ulcer; cancersuch as lung cancer; endotoxin shock; sepsis; and the like. ThePolypeptides of the present invention are in particular of interest inthe context of cardiovascular diseases.

Procedures for Obtaining Polynucleotides of the Present Invention

Polynucleotides of the present invention may be obtained, using standardcloning and screening techniques, from a cDNA library derived from mRNAin cells of human testis tissue, using the expressed sequence tag (EST)analysis (Adams, M. D., et al. Science (1991) 252:1651–1656; Adams, M.D. et al., Nature, (1992) 355:632–634; Adams, M. D., et al., Nature(1995) 377 Supp:3–174). Polynucleotides of the invention can also beobtained from natural sources such as genomic DNA libraries or can besynthesized using well known and commercially available techniques (e.g.F. M. Ausubel et al., 2000, Current Protocols in Molecular Biology).

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of the fusedpolypeptide can be encoded. In certain preferred embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc Natl Acad Sci USA (1989) 86:821–824, or is an HA tag.The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotidesencoding polypeptide variants which comprise one of the amino acidsequences selected from the group of of SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:6, and in which several, for instance from 5 to 10, 1 to 5, 1 to3, 1 to 2 or 1, amino acid residues are substituted, deleted or added,in any combination.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in one of SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5, may be used as hybridization probes for cDNA and genomic DNA oras primers for a nucleic acid amplification (PCR) reaction, to isolatefull-length cDNAs and genomic clones encoding polypeptides of thepresent invention and to isolate cDNA and genomic clones of other genes(including genes encoding paralogs from human sources and orthologs andparalogs from species other than human) that have a high sequencesimilarity to one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. Typicallythese nucleotide sequences are at least 70% identical, preferably atleast 80% and in particular at least 85% identical, more preferably atleast 90% identical, still more preferably at least 95%, still morepreferably at least 96%, still more preferably at least 97%, still morepreferably at least 98%, still more preferably at least 99%, identicalto that of the referent. The probes or primers will generally compriseat least 15 nucleotides, preferably, at least 30 nucleotides and mayhave at least 50 nucleotides. Particularly preferred probes will havebetween 30 and 50 nucleotides. Particularly preferred primers will havebetween 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than human, may beobtained by a process which comprises the steps of screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of one of SEQ ID NO:1, SEQ ID NO:3 orSEQ ID NO:5, or a fragment thereof; and isolating full-length cDNA andgenomic clones containing said polynucleotide sequence. Suchhybridization techniques are well known to the skilled artisan.Preferred stringent hybridization conditions include overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate (w/v), and 20 microgram/mldenatured, sheared salmon sperm DNA; followed by washing the filters in0.1×SSC at about 65° C. Thus the present invention also includespolynucleotides obtainable by screening an appropriate library understringent hybridization conditions with a labeled probe having thesequence of one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or afragment thereof.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide is cut short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low“processivity” (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., PNAS USA 85, 8998–9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an “adaptor”sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the “missing” 5′ end of the cDNA using acombination of gene specific and adaptor specific oligonucleotideprimers. The PCR reaction is then repeated using “nested” primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in the knowngene sequence). The products of this reaction can then be analyzed byDNA sequencing and a full-length cDNA constructed either by joining theproduct directly to the existing cDNA to give a complete sequence, orcarrying out a separate full-length PCR using the new sequenceinformation for the design of the 5′ primer.

Vectors, Host Cells, Expression

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., Molecular Cloning,A Laboratory Manual (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals, i.e.derived from a different species. If a polypeptide of the presentinvention is to be expressed for use in screening assays, it isgenerally possible that the polypeptide be produced at the surface ofthe cell or alternatively in a soluble protein form. If the polypeptideis secreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered. If thepolypeptide is bound at the surface of the cell (membrane boundpolypeptide), usually membrane fractions are prepared in order toaccumulate the membrane bound polypeptide.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and or purification.

Diagnostic Assays

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of thegene characterized by one of the the polynucleotides selected from thegroup of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, which is associatedwith a dysfunction will provide a diagnostic tool that can add to, ordefine, a diagnosis of a disease, or susceptibility to a disease, whichresults from under-expression, over-expression or altered spatial ortemporal expression of the gene. Individuals carrying mutations in thegene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled IGS5 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (ee, e.g., Myers et al.,Science (1985) 230:1242). Sequence changes at specific locations mayalso be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397–4401).

In another embodiment, an array of oligonucleotides probes comprisingIGS5 nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of e.g., genetic mutations. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see for example:M. Chee et al., Science, Vol 274, pp 610–613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to the Diseases through detection of mutation in the IGS5gene by the methods described. In addition, such diseases may bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased level of polypeptide ormRNA. Decreased or increased expression can be measured at the RNA levelusing any of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as a polypeptide of the present invention, ina sample derived from a host are well-known to those of skill in theart. Such assay methods include radio-immuno-assays, competitive-bindingassays, Western Blot analysis and ELISA assays. Thus in another aspect,the present invention relates to a diagnostic kit which comprises:

-   (a) a polynucleotide of the present invention, preferably the    nucleotide sequence of one of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID    NO:5, or a fragment thereof;-   (b) a nucleotide sequence complementary to that of (a);-   (c) a polypeptide of the present invention, preferably the    polypeptide of one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a    fragment thereof; or-   (d) an antibody to a polypeptide of the present invention,    preferably to one of the polypeptides of SEQ ID NO:2, SEQ ID NO:4 or    SEQ ID NO:6.    It will be appreciated that in any such kit, the component (a),    (b), (c) or (d) may constitute a substantial component of said    diagnostic kit. Such a kit will be of use in diagnosing a disease or    susceptibility to a disease, particularly amongst others a disease    as indicated above in the context of the polypeptides of the present    invention.    Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome localization. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thedifferences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Tissue Localization

The nucleotide sequences of the present invention are also valuable fortissue localization. Such techniques allow the determination ofexpression patterns of the IGS5 polypeptides in tissues by detection ofthe mRNAs that encode them. These techniques include in situhybridization techniques and nucleotide amplification techniques, forexample PCR. Such techniques are well known in the art. Results fromthese studies provide an indication of the normal functions of thepolypeptides in the organism. In addition, comparative studies of thenormal expression pattern of IGS5 mRNAs with that of mRNAs encoded by aIGS5 gene provide valuable insights into the role of mutant IGS5polypeptides, or that of inappropriate expression of normal IGS5polypeptides, in disease. Such inappropriate expression may be of atemporal, spatial or simply quantitative nature.

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them, can also be used as immunogens to produceantibodies immunospecific for polypeptides of the present invention. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

Antibodies

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, analogs or cells to an animal, preferably a non-human animal,using routine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495–497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, 77–96, Alan R. Liss, Inc., 1985).Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies. The above-described antibodies maybe employed to isolate or to identify clones expressing the polypeptideor to purify the polypeptides by affinity chromatography. Antibodiesagainst polypeptides of the present invention may also be employed totreat the diseases as indicated above, amongst others.

Fusion Proteins

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises administering to (forexample by inoculation) the mammal a polypeptide of the presentinvention, adequate to produce antibody and/or T cell immune response toprotect said animal from the Diseases hereinbefore mentioned, amongstothers.

Yet another aspect of the invention relates to a method of inducingimmunological response in a mammal which comprises, delivering apolypeptide of the present invention via a vector directing expressionof the polynucleotide and coding for the polypeptide in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a polypeptide of thepresent invention wherein the composition comprises a polypeptide orpolynucleotide of the present invention. Such immunological/vaccineformulations (compositions) may be either therapeuticimmunological/vaccine formulations or prophylactic immunological/vaccineformulations. The vaccine formulation may further comprise a suitablecarrier. Since a polypeptide may be broken down in the stomach, it ispreferably administered parenterally (for instance, subcutaneous,intramuscular, intravenous, or intradermal injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

Polypeptides of the present invention are responsible for one or morebiological functions, including one or more disease states, inparticular the Diseases hereinbefore mentioned. It is therefore desirousto devise screening methods to identify compounds which stimulate orwhich inhibit the function of the polypeptide. Accordingly, in a furtheraspect, the present invention provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the polypeptide. Compounds may be identified from a varietyof sources, for example, cells, cell-free preparations, chemicallibraries, and natural product mixtures. Such stimulators or inhibitorsso-identified may be natural or modified substrates, ligands, receptors,enzymes, etc., as the case may be, of the polypeptide; or may bestructural or functional mimetics thereof (see Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991)).

The screening method may simply measure the influence of a candidatecompound on the activity of the polypeptide, or on cells or membranesbearing the polypeptide. Alternatively, the screening method may involvecompetition with a competitor. Further, these screening methods may testwhether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the activity of the polypeptide or to the cells ormembranes bearing the polypeptide. Inhibition of polypeptide activity isgenerally assayed in the presence of a known substrate and the effect ofthe candidate compound is observed by altered activity, e.g. by testingwhether the candidate compound results in inhibition or stimulation ofthe polypeptide. For example, the screening methods may simply comprisethe steps of mixing a candidate compound with a solution containing apolypeptide of the present invention, and a suitable substrate to form amixture, measuring IGS5 activity in the mixture, and comparing the IGS5activity of the mixture to a standard without candidate compound.

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents which may inhibit or enhancethe production of polypeptide from suitably manipulated cells ortissues. Examples of potential polypeptide inhibitors include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented. Thus, in anotheraspect, the present invention relates to a screening kit for identifyingin particular inhibitors, stimulators, ligands, receptors, substrates,enzymes, etc. for polypeptides of the present invention; or compoundswhich decrease or enhance the production of such polypeptides, whichcomprises:

-   (a) a polypeptide of the present invention;-   (b) a recombinant cell expressing a polypeptide of the present    invention;-   (c) a cell membrane expressing a polypeptide of the present    invention; or-   (d) an antibody to a polypeptide of the present invention; which    polypeptide is preferably one of that of SEQ ID NO:2, SEQ ID NO:4 or    SEQ ID NO:6.    It will be appreciated that in any such kit, the component (a),    (b), (c) or (d) may constitute a substantial part of said kit.

It will be readily appreciated by the skilled artisan that a polypeptideof the present invention may also be used in a method for thestructure-based design of a stimulator or inhibitor of the polypeptide,by:

-   (a) determining in the first instance the three-dimensional    structure of the polypeptide;-   (b) deducing the three-dimensional structure for the likely reactive    or binding site(s) of a stimulator or inhibitor;-   (c) synthesizing candidate compounds that are predicted to bind to    or react with the deduced binding or reactive site; and-   (d) testing whether the candidate compounds are indeed stimulators    or inhibitors.    It will be further appreciated that this will normally be an    iterative process.    Prophylactic and Therapeutic Methods

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, those dysfunctions, disordersor diseases to be treated, hereinabove generally referred to as “thediseases” in the context of the polypeptides of the present invention,related to either an excess of, or an under-expression of IGS5polypeptide activity.

If the activity of the polypeptide is in excess, several approaches areavailable. One approach comprises administering to a subject in needthereof an inhibitor compound as hereinabove described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function of the polypeptide, such as, forexample, by blocking the binding of substrates, enzymes, etc., andthereby alleviating the abnormal condition. In another approach, solubleforms of the polypeptides still capable of binding the substrate,enzymes, etc. in competition with endogenous polypeptide may beadministered. Typical examples of such competitors include fragments ofthe IGS5 polypeptide.

In still another approach, expression of the gene encoding endogenousIGS5 polypeptide can be inhibited using expression blocking techniques.Known such techniques involve the use of antisense sequences, eitherinternally generated or separately administered (see, for example,O'Connor, J. Neurochem. (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). Alternatively, oligonucleotides which form triple helices(“triplexes”) with the gene can be supplied (see, for example, Lee etal., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.Synthetic antisense or triplex oligonucleotides may comprise modifiedbases or modified backbones. Examples of the latter includemethylphosphonate, phosphorothioate or peptide nucleic acid backbones.Such backbones are incorporated in the antisense or triplexoligonucleotide in order to provide protection from degradation bynucleases and are well known in the art. Antisense and triplex moleculessynthesized with these or other modified backbones also form part of thepresent invention.

In addition, expression of the IGS5 polypeptide may be prevented byusing ribozymes specific to the IGS5 mRNA sequence. Ribozymes arecatalytically active RNAs that can be natural or synthetic (see forexample Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527–33.)Synthetic ribozymes can be designed to specifically cleave IGS5 mRNAs atselected positions thereby preventing translation of the IGS5 mRNAs intofunctional polypeptide. Ribozymes may be synthesized with a naturalribose phosphate backbone and natural bases, as normally found in RNAmolecules. Alternatively the ribosymes may be synthesized withnon-natural backbones to provide protection from ribonucleasedegradation, for example, 2′-O-methyl RNA, and may contain modifiedbases.

For treating abnormal conditions related to an under-expression of IGS5and its activity, several approaches are also available. One approachcomprises administering to a subject a therapeutically effective amountof a compound which stimulates a polypeptide of the present invention incombination with a pharmaceutically acceptable carrier, to therebyalleviate the abnormal condition. Alternatively, gene therapy may beemployed to effect the endogenous production of IGS5 by the relevantcells in the subject. For example, a polynucleotide of the invention maybe engineered for expression in a replication defective retroviralvector, as discussed above. The retroviral expression construct may thenbe isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

Formulation and Administration

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide, such as the soluble form of a polypeptide of the presentinvention, stimulating or inhibiting peptide or small molecule compound,in combination with a pharmaceutically acceptable carrier or excipient.Such carriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theinvention further relates to pharmaceutical packs and kits comprisingone or more containers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides and othercompounds of the present invention may be employed alone or inconjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

The dosage range required depends on the choice of peptide or othercompounds of the present invention, the route of administration, thenature of the formulation, the nature of the subject's condition, andthe judgment of the attending practitioner. Suitable dosages, however,are in the range of 0.1–100 μg/kg of subject. Wide variations in theneeded dosage, however, are to be expected in view of the variety ofcompounds available and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject. Polynucleotideand polypeptide sequences form a valuable information resource withwhich it is possible to identify further sequences of similar homology.This is most easily facilitated by storing the sequence in a computerreadable medium and then using the stored data to search a sequencedatabase using well known searching tools, such as those in the GCC andLasergene software packages. Accordingly, in a further aspect, thepresent invention provides for a computer readable medium having storedthereon a polynucleotide comprising the sequences of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5 and/or a polypeptide sequence encoded thereby. Allpublications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The following examples are only intended to further illustrate theinvention, in more detail, and therefore these examples are not deemedto restrict the scope of the invention in any way.

TABLE 1 IGS5-DNA (″IGS5DNA″) of SEQ ID NO:15′-TGCACCACCCCTGGCTGCGTGATAGCAGCTGCCAGGATCCTCCAGAACATGGACCCGACCACGGAACCGTGTGACGACTTCTACCAGTTTGCATGCGGAGGCTGGCTGCGGCGCCACGTGATCCCTGAGACCAACTCAAGATACAGCATCTTTCACGTCCTCCGCGACGAGCTGGAGGTCATCCTCAAAGCGGTGCTGGAGAATTCGACTGCCAAGGACCGGCCGGCTGTGGAGAACGCCAGGACGCTGTACCGCTCCTGCATGAACCAGAGTGTGATAGAGAAGCGAGGCTCTCAGCCCCTGCTGGACATCTTGGAGGTGGTGGGAGGCTGGCCGGTGGCGATGGACAGGTGGAACGAGACCGTAGGACTCGAGTGGGAGCTGGAGCGGCAGCTGGCGCTGATGAACTCACAGTTCAACAGGCGCGTCCTCATCGACCTCTTCATCTGGAACGACGACCAGAACTCCAGCCGGCACATCATCTACATAGACCAGCCCACCTTGGGCATGCCCTCCCGAGAGTACTACTTCAACGGCGGCAGCAACCGGAAGGTGCGGGAAGCCTACCTGCAGTTCATGGTGTCAGTGGCCACGTTGCTGCGGGAGGATGCAAACCTGCCCAGGGACAGCTGCCTGGTGCAGGAGGACATGATGCAGGTGCTGGAGCTGGAGACACAGCTGGCCAAGGCCACCGTACCCCAGGAGGAGAGACACGACGTCATCGCCTTGTACCACCGGATGGGACTGGAGGAGCTGCAAAGCCAGTTTGGCCTGAAGGGATTTAACTCGACTCTGTTCATACAAACTGTGCTATCCTCTGTCAAAATCAAGCTGCTGCCAGATGAGGAAGTGGTGGTCTATGGCATCCCCTACCTGCAGAACCTTGAAAACATCATCGACACCTACTCAGCCAGGACCATACAGAACTACCTGGTCTGGCGCCTGGTGCTGGACCGCATTGGTAGCCTAAGCCAGAGATTCAAGGACACACGAGTGAACTACCGCAAGGCGCTGTTTGGCACAATGGTGGAGGAGGTGCGCTGGCGTGAATGTGTGGGCTACGTCAACAGCAACATGGAGAACGCCGTGGGCTCCCTCTACGTCAGGGAGGCGTTCCCTGGAGACAGCAAGAGCATGGTCAGAGAACTCATTGACAAGGTGCGGACAGTGTTTGTGGAGACGCTGGACGAGCTGGGCTGGATGGACGAGGAGTCCAAGAAGAAGGCGCAGGAGAAGGCCATGAGCATCCGGGAGCAGATCGGGCACCCTGACTACATCCTGGAGGAGATGAACAGGCGCCTGGACGAGGAGTACTCCAATCTGAACTTCTCAGAGGACCTGTACTTTGAGAACAGTCTGCAGAACCTCAAGGTGGGCGCCCAGCGGAGCCTCAGGAAGCTTCGGGAAAAGGTGGACCCAAATCTCTGGATCATCGGGGCGGCGGTGGTCAATGCGTTCTACTCCCCAAACCGAAACCAGATTGTATTCCCTGCCGGGATCCTCCAGCCCCCCTTCTTCAGCAAGCAGCAGCCACAGGCCTTGAACTTTGGAGGCATTGGGATGGTGATCGGGCACGAGATCACGCACGGCTTTGACGACAATGGCCGGAACTTCGACAAGAATGGCAACATGATGGATTGGTGGAGTAACTTCTCCACCCAGCACTTCCGGGAGCAGTCAGAGTGCATGATCTACCAGTACGGCAACTACTCCTGGGACCTGCCAGACGAACAGAACGTGAACGGATTCAACACCCTTGGGGAAAACATTGCTGACAACCGAGGGGTGCGGCAAGCCTATAAGGCCTACCTCAAGTGGATGGCAGAGGGTGGCAAGGACCAGCAGCTGCCCGGCCTGGATCTCACCCATGAGCAGCTCTTCTTCATCAACTACCCCCAGGTGTGGTGCGGGTCCTACCGGCCCGAGTTCGCCATCCAATCCATCAAGACAGACGTCCACAGTCCCCTGAAGTACAGGGTACTGGGGTCGCTGCAGAACCTGGCCGCCTTCGCAGACACGTTCCACTGTGCCCGGGGCACCCCCATGCACCCCAAGGAGCGATGCCGCGTGTGGTAG-3′

TABLE 2 IGS5-protein (″IGS5PROT″) of SEQ ID NO:2CTTPGCVIAAARILQNMDPTTEPCDDFYQFACGGWLRRHVIPETNSRYSIFDVLRDELEVILKAVLENSTAKDRPAVEKARTLYRSCMNQSVIEKRGSQPLLDILEVVGGWPVAMDRWNETVGLEWELERQLALMNSQFNRRVLIDLFIWNDDQNSSRHIIYIDQPTLGMPSREYYFNGGSNRKVREAYLQFMVSVATLLREDANLPRDSCLVQEDMMQVLELETQLAKATVPQEERHDVIALYHRMGLEELQSQFGLKGFNWTLFIQTVLSSVKIKLLPDEEVVVYGIPYLQNLENIIDTYSARTIQNYLVWRLVLDRIGSLSQRFKDTRVNYRKALFGTMVEEVRWRECVGYVNSNMENAVGSLYVREAFPGDSKSMVRELIDKVRTVFVETLDELGWMDEESKKKAQEKAMSIREQIGHPDYILEEMNRRLDEEYSNLNFSEDLYFENSLQNLKVGAQRSLRKLREKVDPNLWIIGAAVVNAFYSPNRNQIVFPAGILQPPFFSKEQPQALNFGGIGMVIGHEITHGFDDNGRNFDKNGNMMDWWSNFSTQHFREQSECMIYQYGNYSWDLADEQNVNGFNTLGENIADNGGVRQAYKAYLKWMAEGGKDQQLPGLDLTHEQLFFINYAQVWCGSYRPEFAIQSIKTDVHSPLKYRVLGSLQNLAAFADTFHCARGTPMHPKERCRVW

TABLE 3 IGS5-DNA-1 (″IGS5DNA1″) of SEQ ID NO:35′-ATGGGGAAGTCCGAAGGCCCCGTGGGGATGGTGGAGAGCGCTGGCCGTGCAGGGCAGAAGCGCCCGGGGTTCCTGGAGGGGGGGCTGCTGCTGCTGCTGCTGCTGGTGACCGCTGCCCTGGTGGCCTTGGCTGTCCTCTACGCCGACCGCAGAGGGAAGCACCTGCCACGCCTTGCTAGCCGGCTGTGCTTCTTACAGGAGGAGAGGACCTTTGTAAAACGAAAACCCCGAGGGATCCCAGAGGCCCAAGAGGTGAGCGAGGTCTGCACCACCCCTGGCTGCGTGATAGCAGCTGCCAGGATCCTCCAGAACATGGACCCGACCACGGAACCGTGTGACGACTTCTACCAGTTTGCATGCGGAGGCTGGCTGCCGCGCCACGTGATCCCTGAGACCAACTCAAGATACAGCATCTTTGACGTCCTCCGCGACGAGCTGGAGGTCATCCTCAAAGCGGTGCTGGAGAATTCGACTGCCAAGGACCGGCCGGCTGTGGAGAAGGCCAGGACGCTGTACCGCTCCTGCATGAACCAGAGTGTGATAGAGAAGCGAGGCTCTCAGCCCCTGCTGGACATCTTGGAGGTGGTGGGAGGCTGGCCGGTGGCGATGGACACGTGGAACGACACCGTAGGACTCCAGTGGGAGCTCGACCCCCACCTCGCGCTGATGAACTCACAGTTCAACAGGCGCGTCCTCATCGACCTCTTCATCTGGAACGACGACCAGAACTCCAGCCGGCACATCATCTACATAGACCAGCCCACCTTGGGCATGCCCTCCCGAGAGTACTACTTCAACGGCGGCAGCAACCGGAAGGTGCGGGAAGCCTACCTCCACTTCATGGTGTCAGTGGCCACCTTGCTGCGGGACGATGCAAACCTGCCCAGGGACAGCTGCCTGGTGCAGGAGGACATGATGCAGGTGCTGGAGCTGGAGACACAGCTGGCCAAGGCCACGGTACCCCAGGAGGAGAGACACGACGTCATCGCCTTGTACCACCGGATGGGACTGGAGGAGCTGCAAAGCCAGTTTGGCCTCAAGGGATTTAACTCGACTCTCTTCATACAAACTCTGCTATCCTCTGTCAAAATCAACCTGCTGCCACATCACCAACTCCTCCTCTATCCCATCCCCTACCTCCAGAACCTTGAAAACATCATCGACACCTACTCAGCCAGGACCATACAGAACTACCTGGTCTGGCGCCTGGTGCTGGACCGCATTGGTAGCCTAAGCCAGAGATTCAAGGACACACGAGTGAACTACCCQAAGGCGCTGTTTGGCACAATGGTGGAGGAGGTGCGCTGGCGTGAATCTGTGGCCTACGTCAACAGCAACATGGAGAACGCCGTGGGCTCCCTCTACGTCAGCGACCCCTTCCCTGGAGACAGCAAGACCATGGTCACAGAACTCATTGACAACGTGCGGACAGTGTTTGTGGAGACGCTGGACGAGCTGGGCTGGATGGACGAGGAGTCCAAGAAGAAGGCGCAGGAGAAGGCCATCAGCATCCGGGAGCAGATCCCGCACCCTGACTACATCCTGGAGGAGATGAACAGGCGCCTGCACGAGGACTACTCCAATCTCAACTTCTCAGAGGACCTGTACTTTCACAACAGTCTCCACAACCTCAAGGTGGCCGCCCACCGCAGCCTCAGCAAGCTTCCGGAAAAGCTGGACCCAAATCTCTCCATCATCGGCGCGCCGGTGGTCAATGCGTTCTACTCCCCAAACCGAAACCACATTCTATTCCCTCCCCCCATCCTCCACCCCCCCTTCTTCACCAACCACCACCCACAGCCCTTGAACTTTGCACCCATTCCCATCGTCATCGGGCACGAGATCACGCACGGCTTTGACGACAATGGCCGGAACTTCGACAAGAATGGCAACATGATGGATTGGTGGAGTAACTTCTCCACCCACCACTTCCGGGAGCAGTCAGAGTGCATGATCTACCAGTACCCCAACTACTCCTGGCACCTCCCAGACCAACACAACGTGAACGGATTCAACACCCTTGCCCAAAACATTCCTGACAACCCACCGGTGCGGCAAGCCTATAACGCCTACCTCAAGTGGATGGCAGACGGTGGCAAGGACCAGCAGCTGCCCGGCCTGGATCTCACCCATGAGCAGCTCTTCTTCATCAACTACGCCCAGGTGTGGTGCGGGTCCTACCGGCCCGAGTTCGCCATCCAATCCATCAACACACACGTCCACACTCCCCTCAACTACACGGTACTCCGCTCCCTGCACAACCTCCCCCCCTTCCCAGACACGTTCCACTGTGCCCGGGGCACCCCCATGCACCCCAAGGAGCGATGCCGCGTGTGGTAG-3′

TABLE 4 IGS5-protein-1 (″IGS5PROT1″) of SEQ ID NO:4MGKSEGPVGMVESAGPAGQKRPGFLEGGLLLLLLLVTAALVALGVLYADRRGKQLPRLASRLCFLQEERTFVKRKPRGIPEAQEVSEVCTTPGCVTAAARILQNMDPTTEPCDDFYQFACGGWLRRHVTPETNSRYSIFDVLRDELEVTLKAVLENSTAKDRPAVEKARTLYRSCMNQSVIEKRGSQPLLDILEVVGGWPVAMDRWNETVGLEWELERQLALMNSQFNRRVLIDLFIWNDDQNSSRHIIYIDQPTLGMPSREYYFNGGSNRKVREAYLQFMVSVATLLREDANLPRDSCLVQEDMMQVLELETQLAKATVPQEERHDVIALYHRMGLEELQSQFGLKGFNWTLFIQTVLSSVKIKLLPDEEVVVYGIPYLQNLENIIDTYSARTIQNYLVWRLVLDRIGSLSQRFKDTRVNYRKALFGTMVEEVRWRECVGYVNSNMENAVGSLYVREAFPGDSKSMVRELIDKVRTVFVETLDELGWMDEESKKKAQEKAMSTREQTGHPDYILEEMNRRLDEEYSNLNFSEDLYFENSLQNLKVGAQRSLRKLREKVDPNLWIIGAAVVNAFYSPNRNQIVFPAGILQPPFFSKEQPQALNFGGIGMVIGHEITHGFDDNGRNFDKNGNMMDWWSNFSTQHFREQSECMIYQYGNYSWDLADEQNVNGFNTLGENIADNGGVRQAYKAYLKWMAEGGKDQQLPGLDLTHEQLFFINYAQVWCGSYRPEFAIQSIKTDVHSPLKYRVLGSLQNLAAFADTFHCARGTPMHPKERCRVW

TABLE 5 IGSS-DNA-2 (″IGS5DNA2″) of SEQ ID NO:55′-ATGGGGAAGTCCGAAGGCCCAGTGGGGATGGTGGAGAGCGCCGGCCGTGCAGGGCAGAAGCGCCCGGGGTTCCTGGAGGGGGGGCTGCTGCTGCTGCTGCTGCTGGTGACCGCTGCCCTGGTGGCCTTGGGTGTCCTCTACGCCGACCGCAGAGGGATCCCAGAGGCCCAAGAGGTGAGCGAGGTCTGCACCACCCCTGGCTGCGTGATAGCAGCTGCCAGGATCCTCCAGAACATGGACCCGACCACGGAACCGTGTGACGACTTCTACCAGTTTGCATGCGGAGGCTGGCTGCGGCGCCACGTGATCCCTGAGACCAACTCAAGATACAGCATCTTTGACGTCCTCCGCGACGAGCTGGAGGTCATCCTCAAAGCGGTGCTGGAGAATTCGACTGCCAAGGACCGGCCGGCTGTGGAGAAGGCCAGGACGCTGTACCGCTCCTGCATGAACCAGAGTGTGATAGAGAAGCGAGGCTCTCAGCCCCTGCTGGACATCTTGGAGGTGGTGGGAGGCTGGCCGGTGGCGATGGACAGGTGGAACGAGACCGTAGGACTCGAGTGGGAGCTGGAGCGGCAGCTGGCGCTGATGAACTCACAGTTCAACACCCCCGTCCTCATCGACCTCTTCATCTGGAIACCACCACCACACTCCACCCGCCACATCATCTACATAGACCAGCCCACCTTGGGCATGCCCTCCCGAGAGTACTACTTCAACGGCGGCAGCAACCGGAAGGTGCGGGAAGCCTACCTGCAGTTCATGGTGTCAGTGGCCACGTTGCTGCGGGAGGATGCAAACCTGCCCAGGGACAGCTCCCTCGTGCACGAGGACATGATGCAGGTGCTGGAGCTCGACACACAGCTGGCCAAGGCCACGGTACCCCAGGAGGAGAGACACGACGTCATCGCCTTGTACCACCGGATGGGACTGCAGGAGCTGCAAAGCCAGTTTGGCCTGAAGGGATTTAACTGGACTCTGTTCATACAAACTGTGCTATCCTCTGTCAAAATCAAGCTCCTGCCACATCAGGAAGTGGTGGTCTATGGCATCCCCTACCTGCAGAACCTTGAAAACATCATCGACACCTACTCAGCCAGGACCATACAGAACTACCTGGTCTGGCGCCTGGTGCTGGACCGCATTGGTAGCCTAAGCCAGAGATTCAAGGACACACGAGTGAACTACCGCAAGGCGCTGTTTGGCACAATCCTCGAGGAGGTGCGCTGGCGTGAATCTGTCCCCTACCTCAACAGCAACATGGAGAACGCCGTCCCCTCCCTCTACGTCACGGACCCGTTCCCTGGACACAGCAAGAGCATGGTCAGAGAACTCATTGACAAGGTGCGGACAGTGTTTGTGGAGACGCTGGACGAGCTGGGCTGGATGCACGAGGAGTCCAAGAAGAAGGCGCAGGAGAAGGCCATGAGCATCCGGGAGCAGATCCCGCACCCTCACTACATCCTGGAGGACATCAACAGCCCCCTGGACGAGGAGTACTCCAATCTGAACTTCTCAGAGGACCTGTACTTTGAGAACAGTCTGCAGAACCTCAAGGTGGGCGCCCAGCGGAGCCTCAGGAAGCTTCGGGAAAAGGTGGACCCAAATCTCTGGATCATCGGGGCGGCGGTGCTCAATGCGTTCTACTCCCCAAACCGAAACCAGATTGTATTCCCTGCCGGGATCCTCCAGCCCCCCTTCTTCACCAAGGAGCAGCCACACCCCTTCAACTTTGGAGGCATTGGGATGGTGATCGGGCACGAGATCACGCACGGCTTTGACGACAATGGCCCGAACTTCGACAAGAATGGCAACATCATGGATTGGTGGACTAACTTCTCCACCCAGCACTTCCGGGAGCAGTCAGAGTGCATGATCTACCACTACGGCAACTACTCCTGGCACCTGGCAGACGAACAGAACGTGAACGCATTCAACACCCTTGCGGAAAACATTCCTGACAACGGAGCCCTCCGGCAAGCCTATAAGGCCTACCTCAAGTGGATGGCAGAGGGTGGCAAGGACCAGCAGCTGCCCGGCCTGGATCTCACCCATGAGCAGCTCTTCTTCATCAACTACGCCCAGGTGTGGTGCGGGTCCTACCGGCCCGAGTTCGCCATCCAATCCATCAAGACAGACGTCCACAGTCCCCTGAAGTACAGGGTACTCGCGTCGCTGCACAACCTGGCCGCCTTCGCACACACGTTCCACTGTGCCCGGGGCACCCCCATGCACCCCAAGGAGCCA TCCCGCGTGTGGTAG-3′

TABLE 6 IGS5-protein-2 (″IGS5PROT2″) of SEQ ID NO:6MGKSEGPVGMVESAGRAGQKRPGFLEGGLLLLLLLVTAALVALGVLYADRRGIPEAQEVSEVCTTPGCVTAAARTLQNMDPTTEPCDDFYQFACGGWLRRHVIPETNSRYSTFDVLRDELEVILKAVLENSTAKDRPAVEKARTLYRSCMNQSVIEKRGSQPLLDILEVVGGWPVANDRWNETVGLEWELERQLALMNSQFNRRVLIDLFTWNDDQNSSRHIIYIDQPTLGMPSREYYFNGGSNRKVREAYLQFMVSVATLLREDANLPRDSCLVQEDMMQVLELETQLAKATVPQEERHDVIALYHRMGLEELQSQFGLKGFNWTLFIQTVLSSVKIKLLPDEEVVVYGIPYLQNLENIIDTYSARTIQNYLVWRLVLDRTGSLSQRFKDTRVNYRKALFGTMVEEVRWRECVGYVNSNMENAVGSLYVREAFPGDSKSMVRELIDKVRTVFVETLDELGWMDEESKKKAQEKAMSIREQIGHPDYTLEEMNRRLDEEYSNLNFSEDLYFENSLQNLKVGAQRSLRKLREKVDPNLWIIGAAVVNAFYSPNRNQIVFPAGILQPPFFSKEQPQALNFGGIGMVIGHEITHGFDDNGRNFDKNGNMMDWWSNFSTQHFREQSECMIYQYGNYSWDLADEQNVNGFNTLGENIADNGGVRQAYKAYLKWMAEGGKDQQLPGLDLTHEQLFFINYAQVWCGSYRPEFAIQSIKTDVHSPLKYRVLGSLQNLAAFADTFHCAIRGTRMHPKERC RVW

EXAMPLE 1

The Cloning of cDNA Encoding a Novel Member of the NEP/ECEMetalloprotease Family

Example 1a

Homology PCR Cloning of a cDNA Fragment of a Novel Member of the NEP/ECEMetalloprotease Family

In the DNA databank of expressed sequence tags (ESTs) 4 overlapping ESTsequences (accession nos. AA524283, AI088893, AI217369 and AI380811)were detected which contained a small open reading frame encoding astretch of protein that showed similarity to the C-terminal part ofmembers of the neutral endopeptidase 24.11/endothelin converting enzyme(NEP/ECE) metalloprotease protein family (Turner A. J. et al. Faseb J.(1997) 11: 355–364). The NEP/ECE-like small open reading frame in theseESTs was terminated by a stop codon (in the case of AA524283) and waspreceded in all 4 ESTs by a sequence that contained stop codons in all 3reading frames. This preceeding sequence appeared totally unrelated toNEP/ECE metalloprotease family members.

Although the polarity of the small open reading frame was opposite tothe 5′.fwdarw.3′ orientation of the mRNA from which these ESTs had beenderived, these sequences were used as the basis for a RT-PCR homologycloning approach. In parallel, additional EST sequences, that showed thesame structure as the 4 ESTs mentioned before, were observed to appearin the public domain databanks, e.g. accession nos: AI825876, AI888306,AI422224, AI422225, AI469281, AA975272, AA494534, AW006103, AI827701,AI650385, AI827898, AI934499 and AA422157.

The RT-PCR reactions were carried out using a reverse primer (IP11689;SEQ ID NO:7) designed on the EST cluster (within the area showingsimilarity to the NEP/ECE family) and a degenerated forward primer(IP11685; SEQ ID NO:8), centered on a conserved peptide motif(VNA(F,Y)Y) of the NEP/ECE family.

For the synthesis of cDNA 2 μg human lung total RNA (Clontech #64023–1),1 μl oligo(dT)₁₂₋₁₈ (500 μg/ml) and 9 μl H₂O were combined (finalvolume=12 μl), heated to 70° C. for 10 minutes and then chilled on ice.4 μl 5× first strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mMMgCl₂), 2 μl 0.1M DTT, 1 μl 10 mM dNTP mix and 1 μl (200 U) Superscript™II (Life Technologies) reverse transcriptase were added. The mixture wasincubated at 42° C. for 50 minutes and the reaction was inactivated byheating at 70° C. for 15 minutes.

The PCR reaction was performed in a 50 μl volume containing 1 μl of thecDNA synthesis reaction, 5 μl of GeneAmp™ 10×PCR buffer (500 mM KCl, 100mM Tris pH 8.3, 15 mM MgCl₂, 0.01% (w/v) gelatin; Perkin Elmer), 2 μl of10 mM dNTP mix, 10 pmoles each of the forward and reverse primers and 5units AmpliTaq™ polymerase (Perkin Elmer). After an initial denaturationat 95° C. for 5 min., PCR reactions were cycled 40× as follows: 1 mindenaturation at 94° C., 1 min annealing at 60° C. and 1 min extension at72° C. PCR reaction products were analyzed by agarose gelelectrophoresis.

The IP11685/IP11689 RT-PCR reaction produced an amplicon of .+-.600 basepairs (bp). The fragment was purified from gel using the Qiaex-II™purification kit (Qiagen) and ligated into the pGEM-T Easy plasmidaccording to the procedure recommended by the supplier (pGEM-T Easysystem, Promega). The recombinant plasmids were then used to transformcompetent E. coli SURE™ 2 bacteria (Stratagene). Transformed cells wereplated on LB agar plates containing ampicillin (100 μg/I), IPTG (0.5 mM)and X-gal (50 μg/ml). Plasmid DNA was purified from mini-cultures ofindividual colonies using the BioRobot™ 9600 nucleic acid purificationsystem (Qiagen).

DNA Sequencing reactions were carried out on the purified plasmid DNAwith the ABI Prism™ BigDye™ Terminator Cycle Sequencing Ready Reactionkit (PE-ABI), using insert-flanking or internal (IGS5 specific) primers.Plasmid inserts were completely sequenced on both strands. CycleSequencing reaction products were purified via EtOH/NaOAc precipitationand analyzed on an ABI 373 automated sequencer. The DNA sequence of theinserts of recombinant clones YCE14, YCE15 and YCE16 (derived from theIP11685/IP11689 amplicon) extended the open reading frame of theoriginal EST cluster in the direction of the N-terminus and furtherconfirmed that this open reading frame was derived from a novel memberof the NEP/ECE metalloprotease protein family (see FIG. 1). Thisupstream sequence thus deviated completely from the upstream sequencepresent in the EST sequences. This novel sequence is referred to withinthe context of the present invention generally as “IGS5.”

Example 1b

Cloning of cDNA Containing the Putative Ectodomain of IGS5

In order to obtain additional IGS5 cDNA sequence another round of RT-PCRreactions were carried out on human lung RNA under the conditionsdescribed above using the IGS5 specific reverse primer IP12190 (SEQ IDNO:9) and a degenerated forward primer (IP12433; SEQ ID NO:10), centeredon a conserved peptide motif (LXXLXWMD) of the NEP/ECE family. TheIP12190/12433 RT-PCR reaction produced an amplicon of .+−.600 bp thatwas cloned into the pGEM-T Easy vector yielding clones YCE19, YCE22 andYCE23. All clones were fully sequenced and allowed to extend the IGS5open reading frame further upstream (see FIG. 1). To obtain cDNA clonesthat would cover the 5′ end of the IGS5 transcript, semi-nested 5′-RACEPCR reactions were done on human heart Marathon-Ready™ cDNA using theadaptor primer 1 (AP1: SEQ ID NO:11) provided with the Marathon™ cDNAamplification kit (Clontech K1802–1) in combination with IGS5 specificprimers IP12189 SEQ ID NO:12) and IP12585 (SEQ ID NO:13). PCR RACEreactions were performed according to the instructions of theMarathon-Ready™ cDNA user manual provided by Clontech. RACE productswere separated on agarose gel, visualized with ethidium bromide andblotted onto Hybond N⁺ membranes. Blots were prehybridized at 65° C. for2 h in modified Church buffer (0.5 M phosphate, 7% SDS, 10 mM EDTA) andthen hybridized overnight at 65° C. in the same buffer containing 2×10⁶cpm/ml of the ³²P-labelled insert of clone YCE23. The YCE23 insert wasradiolabelled via random primed incorporation of (α-³²P)dCTP to aspecific activity of >10⁹ cpm/μg using the Prime-It II™ kit (Stratagene)according to the instructions provided by the supplier. Hybridized blotswere washed at high stringency (2×30 min at room temperature in2×SSC/0.1% SDS followed by 2 washes of 40 min at 65° C. in 0.1×SSC, 0.1%SDS) and autoradiographed overnight. Hybridizing fragments were purifiedfrom gel, cloned into the pGEM-T Easy vector (yielding clones YCE 59,YCE 64 and YCE 65) and sequenced as described above.

The DNA sequences of all isolated clones could be assembled into asingle contig (IGS5CONS; see FIG. 1) that extended the open readingframe of IGS5 further upstream but an ATG start of translation codon wasnot yet encountered. Primer IP11689 had been designed on EST AI380811and did not contain the last 4 nucleotides before the stop codon presentin the aligned EST sequences. In order to generate an open reading framethat terminated at the stop codon the last (consensus) 22 nucleotides ofthe aligned EST sequences were included in the overall assembly ofIGS5CONS.

Homology searches showed that the (partial) encoded protein was mostsimilar to neutral endopeptidase (NEP; see example 2). However, theinitial 20 amino acids of the IGS5CONS open reading frame did not showany similarity to NEP. This could possibly be due to the fact that theywere derived from an intron. Indeed exon 4 of human NEP starts at aposition that corresponds approximately to the position downstream ofthese 20 amino acids (D'Adamio L. et al. Proc. Natl. Acad. Sci. USA(1989) 86: 7103–7107). Hydropathy analysis (Kyte J. et al. (1982) J.Mol. Biol. 157: 105–132; Klein P. et al. (1985) Biochim. Biophys. Acta815:468–476) did not indicate the presence of a transmembrane domainwithin the predicted IGS5CONS amino acid sequence, although such atransmembrane domain would be expected to occur (or at least overlapwith) within the initial 20 amino acids. For these reasons it waspreferred to exclude the initial sequence part of the IGS5 contig (FIG.1). The resulting DNA sequence (IGS5DNA; SEQ ID NO:1) is 2076nucleotides long (including the stop codon) and encodes a protein of 691residues (IGS5PROT, SEQ ID NO:2). Alignment of IGS5PROT with the humanNEP protein sequence showed that the IGS5PROT sequence corresponds tothe complete ectodomain sequence of NEP. IGS5PROT can thus be expectedto carry the complete enzymatic activity of the putative IGS5 enzyme, aswas demonstrated for the ectodomain of NEP (Fossiez F. et al. Biochem.J. (1992) 284, 53–59).

TABLE 7 Overview of oligo primers used in Example 1 SEQ ID NO:7 IP11689:5′-ACACGGCATCGCTCCTTC-3′ SEQ ID NO:8 IP11685: 5′-CCCCCTGGACGGTGAA (C orT) GC (A, C, G or T) T (A or T) (C or T) TA-3′ SEQ ID NO:9 IP12190:5′-AATCCGTTCACGTTCTGTTCGTCTGCC-3′ SEQ ID NO:10 IP12433: 5′-CCTGGAGGAGCTG(A, C or C) (A, C or T) (A, C, G or T) TGGATG (A or G) A-3′ SEQ ID NO:11AP1: 5′-CCATCCTAATACGACTCACTATAGGGC-3′ SEQ ID NO:12 IP12189:5′-GTCCTTGCCACCCTCTGCCATCC-3′ SEQ ID NO:13 IP12585:5′-ACCACCCCCGCCCCGATGATCCAGAG-3′

EXAMPLE 2

Alignment of IGS5 with Protein Sequences of Members of the NEP/ECEMetalloprotease Family

For the IGS5 Sequence cloned in example 1a, homology searches of up todate protein databanks and translated DNA databanks were executed usingthe BLAST algorithm (Altschul S. F. et al. (1997), Nucleic Acids Res.25:3389–3402). These searches showed that the IGS5 protein was mostsimilar (54–55% identities over .+−. 700 aligned residues) to mouse, ratand human neutral endopeptidase (SW:NEP_MOUSE, accession no. Q61391;SW:NEP_RAT, accession no. P07861 and SW:NEP_HUMAN accession no. P08473).

Thus, this alignment of the almost complete IGS5 protein sequence withthe other members of the NEP/ECE family shows the relation of IGS5 tometalloproteases in general, and in particular to the NEP and/or ECEmetalloprotease families. From this structural alignment it is concludedthat the IGS5 has the functionality of metalloproteases, which in turnare of interest in the context of several dysfunctions, disorders ordiseases in animals and humans.

EXAMPLE 3

The Cloning of cDNA Encoding a Novel Member of the NEP/ECEMetalloprotease Family

Example 3a

Homology PCR Cloning of a cDNA Fragment of a Novel Member of the NEP/ECEMetalloprotease Family

In the DNA databank of expressed sequence tags (ESTs) 4 overlapping ESTsequences (accession nos. AA524283, AI088893, AI217369 and AI380811)were detected which contained a small open reading frame encoding astretch of protein that showed similarity to the C-terminal part ofmembers of the neutral endopeptidase 24.11/endothelin converting enzyme(NEP/ECE) metalloprotease protein family (Turner A. J. et al., Faseb J.(1997) 11: 355–364). The NEP/ECE-like small open reading frame in theseESTs was terminated by a stop codon (in the case of AA524283) and waspreceded in all 4 ESTs by a sequence that contained stop codons in all 3reading frames. This preceding sequence appeared totally unrelated toNEP/ECE metalloprotease family members. Although the polarity of thesmall open reading frame was opposite to the 5′.fwdarw.3′ orientation ofthe mRNA from which these ESTs had been derived, it was decided to usethese sequences as the basis for a RT-PCR homology cloning approach. Inparallel, additional EST sequences, that showed the same structure asthe 4 ESTs mentioned before were observed to appear in the public domaindatabanks, e.g. accession nos: AI825876, AI888306, AI422224, AI422225,AI469281, AA975272, AA494534, AW006103, AI827701, AI650385, AI827898,AI934499 and AA422157. The RT-PCR reactions were carried out using areverse primer (IP11689; SEQ ID NO:7) designed on the EST cluster(within the area showing similarity to the NEP/ECE family) and adegenerated forward primer (IP11685; SEQ ID NO:8), centered on aconserved peptide motif (VNA(F,Y)Y) of the NEP/ECE family.

For the synthesis of cDNA 2 μg human lung total RNA (Clontech #64023–1),1 μl oligo(dT)₁₂₋₁₈ (500 μg/ml) and 9 μl H₂O were combined (finalvolume=12 μl), heated to 70° C. for 10 minutes and then chilled on ice.4 μl 5× first strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mMMgCl₂), 2 μl 0.1 M DTT, 1 μl 10 mM dNTP mix and 1 μl (200 U)Superscript™ II (Life Technologies) reverse transcriptase were added.The mixture was incubated at 42° C. for 50 minutes and the reaction wasinactivated by heating at 70° C. for 15 minutes.

The PCR reaction was performed in a 50 μl volume containing 1 μl of thecDNA synthesis reaction, 5 μl of GeneAmp™ 10×PCR buffer (500 mM KCl, 100mM Tris pH 8.3, 15 mM MgCl₂, 0.01% (w/v) gelatin; PE Biosystems), 2 μlof 10 mM dNTP mix, 10 pmoles each of the forward and reverse primers and5 units AmpliTaq™ polymerase (PE Biosystems). After an initialdenaturation at 95° C. for 5 min., PCR reaction tubes were cycled 40× asfollows: 1 min denaturation at 94° C., 1 min annealing at 60° C. and 1min extension at 72° C. PCR reaction products were analyzed by agarosegel electrophoresis.

The IP11685/IP11689 RT-PCR reaction produced an amplicon of .+-.600 basepairs (bp). The fragment was purified from gel using the Qiaex-II™purification kit (Qiagen) and ligated into the pGEM™-T Easy plasmidaccording to the procedure recommended by the supplier (pGEM™-T Easysystem, Promega). The recombinant plasmids were then used to transformcompetent E. coli SURE™ 2 bacteria (Stratagene). Transformed cells wereplated on LB agar plates containing ampicillin (100 μg/ml), IPTG (0.5mM) and X-gal (50 μg/ml). Plasmid DNA was purified from mini-cultures ofindividual colonies using the BioRobot™ 9600 nucleic acid purificationsystem (Qiagen).

DNA sequencing reactions were carried out on the purified plasmid DNAwith the ABI Prism™ BigDye™ Terminator Cycle Sequencing Ready Reactionkit (PE Biosystems), using insert-flanking or internal primers. Plasmidinserts were completely sequenced on both strands. Cycle Sequencingreaction products were purified via EtOH/NaOAc precipitation andanalyzed on an ABI 377 automated sequencer. The DNA sequence of theinserts of recombinant clones YCE14, YCE15 and YCE16 (derived from theIP11685/IP11689 amplicon) extended the open reading frame of theoriginal EST cluster in the direction of the N-terminus and furthersupported the hypothesis that this open reading frame was derived from anovel member of the NEP/ECE metalloprotease protein family (FIG. 2).This upstream sequence thus deviated completely from the upstreamsequence present in the EST sequences. This novel sequence is referredto within the context of the present invention generally as “IGS5.”

Example 3b

Cloning of cDNA Fragments Containing the Full Length Coding Sequence ofIGS5

In order to obtain additional IGS5 cDNA sequence another round of RT-PCRreactions were carried out on human lung RNA under the conditionsdescribed above using the IGS5 specific reverse primer IP12190 (SEQ IDNO:9) and a degenerated forward primer (IP12433; SEQ ID NO:10), centeredon a conserved peptide motif (LXXLXWMD) of the NEP/ECE protein family.The IP12190/12433 RT-PCR reaction produced an amplicon of .+−.600 bpthat was cloned into the pGEM™-T Easy vector yielding clones YCE19,YCE22 and YCE23. All clones were fully sequenced and allowed to extendthe IGS5 open reading frame further upstream (see FIG. 2).

To obtain cDNA clones that would cover the 5′ end of the IGS5transcript, semi-nested 5′-RACE PCR reactions were done on human heartMarathon-Ready™ cDNA using the adaptor primer 1 (AP1: SEQ ID NO:11)provided with the Marathon™ cDNA amplification kit (Clontech K1802–1) incombination with IGS5 specific primers IP12189 (SEQ ID NO:12) andIP12585 (SEQ ID NO13). PCR RACE reactions were performed according tothe instructions of the Marathon-Ready™ cDNA user manual provided byClontech. RACE products were separated on agarose gel, visualized withethidium bromide and blotted onto Hybond™-N⁺ membranes (Amersham). Blotswere prehybridized at 65° C. for 2 h in modified Church buffer (0.5 Mphosphate, 7% SDS, 10 mM EDTA) and then hybridized overnight at 65° C.in the same buffer containing 2×10⁶ cpm/ml of the ³²P-labelled insert ofclone YCE23. The YCE23 insert was radiolabelled via random primedincorporation of (α-³²P)dCTP to a specific activity of >10⁹ cpm/μg usingthe Prime-It II™ kit (Stratagene) according to the instructions providedby the supplier. Hybridized blots were washed at high stringency (2×30min at room temperature in 2×SSC/0.1% SDS followed by 2 washes of 40 minat 65° C. in 0.1×SSC, 0.1% SDS) and autoradiographed overnight.Hybridizing fragments were purified from gel, cloned into the pGEM™-TEasy vector (yielding clones YCE 59, YCE 64 and YCE 65) and sequenced asdescribed above.

The DNA sequences of all isolated clones could be assembled into asingle contig that extended the open reading frame of IGS5 furtherupstream although no start of translation codon was yet encountered.Primer IP11689 had been designed on EST AI380811 and did not incorporatethe last 4 nucleotides before the stop codon present in the aligned ESTsequences. In order to generate an open reading frame that terminated atthis stop codon the last (consensus) 22 nucleotides of the aligned ESTsequences were included in the contig.

Several attempts to clone the still missing amino-terminal part of theIGS5 coding sequence via 5′ RACE PCR extension or via screening of cDNAlibraries failed. Therefore it was tried to obtain genomic sequenceinformation in the area around and upstream of the 5′ end of thepreliminary IGS5 contig. Approximately 550,000 plaques of a humangenomic DNA library, constructed in the lambda EMBL3 phage vector(Clontech HL1067j) were lifted onto Hybond™-N⁺ membranes. Membrane liftswere prehybridized at 65° C. for 2 h in modified Church buffer and thenhybridized overnight at 65° C. in the same buffer containing 2×10⁶cpm/ml of a ³²P-labeled .+−. 150 bp EcoRI/EcoRII fragment, located atthe 5′ end of clone YCE59. The cDNA probe was radiolabelled via randomprimed incorporation of (α-³²P)dCTP to a specific activity of >10⁹cpm/μg using the Prime-It II kit™ (Stratagene) according to theinstructions provided by the supplier. Hybridized membranes were washedat high stringency (2×30 min at room temperature in 2×SSC/0.1% SDSfollowed by 1 wash of 40 min at 65° C. in 0.1×SSC/0.1% SDS) andautoradiographed. Hybridizing plaques were subjected to a second roundof screening and pure single plaques were obtained. Recombinant phageDNA was purified from infected liquid cultures using the Qiagen™ LambdaMidi Kit (Qiagen) and sequenced as described above using flanking EMBL3vector primers and IGS5 internal primers. From the insert of cloneIGS5/S1 approximately 5,000 nucleotides upstream of the 5′ end of thepreliminary IGS5 contig were sequenced. Homology searches of translatedDNA databanks showed that this 5,000 bp fragment contained a stretch of78 bp which encoded a peptide that was most similar (15 identicalresidues over 25 aligned) to an alternatively spliced 69 bp fragment inthe mouse SEP sequence (GenBank accession no AF157105), which is arecently described novel member of the NEP/ECE family (Ikeda et al.(1999) JBC 274: 32469–32477). This 78 bp human fragment was preceded byand followed by putative consensus splice acceptor and donor sitesrespectively but did not contain an “ATG” start of translation codon.

In order to obtain cDNA clones containing the amino-terminal part of theIGS5 coding sequence, semi-nested 5′ RACE PCR reactions were carried outon human testis Marathon-Ready™ cDNA (Clontech 7414–1) using the adapterprimer 1 (AP1: SEQ ID NO:11) provided with the Marathon™ cDNAamplification kit (Clontech K1802–1) in combination with IGS5 specificanti-sense primers IP14,241 (SEQ ID NO:14) and IP14242 (SEQ ID NO:15)which were designed within the 78 bp genomic fragment described above.PCR RACE reactions were performed according to the instructions of theMarathon Ready™ cDNA user manual provided by Clontech (reactionvolume=25 μl). RACE products were separated on agarose gel, visualisedwith ethidium bromide and analyzed via Southern blot.

To generate a specific hybridizaton probe for the blotted RACE products,a semi-homology PCR reaction was carried out on the above obtainednested RACE products using the reverse oligonucleotide primer IP14241(SEQ ID NO:14) and a degenerated forward primer (IP13798; SEQ ID NO:16)which was centered on a peptide motif (GLMVLLLL) within thetransmembrane domain of the mouse SEP protein. The PCR reaction wasperformed in a 25 μl volume containing 1 μl of the semi-nested 5′ RACEPCR reaction product, 2.5 μl of GeneAmp™ 10×PCR buffer (500 mM KCl, 100mM Tris pH 8.3, 15 mM MgCl₂, 0.01% (w/v) gelatin; PE Biosystems), 1 μlof 10 mM dNTP mix, 10 pmoles each of the forward and reverse primers and2.5 units AmpliTaq-Gold™ polymerase (PE Biosystems). After an initialdenaturation at 95° C. for 10 min, PCR reaction tubes were cycled 35× asfollows: 1 min denaturation at 95° C., 30 seconds annealing at 50° C.and 30 seconds extension at 72° C. PCR reaction products were analyzedvia agarose gel electrophoresis. The semi-homology PCR reaction producedan amplicon of .+−.110 base pairs. The fragment was purified from gelusing the Qiaex II™ purification kit (Qiagen) and ligated into thepGEM™-T plasmid according to the procedure recommended by the supplier(pGEM™-T system, Promega). The recombinant plasmids were then used totransform competent E. coli SURE™ 2 bacteria (Stratagene). Transformedcells were plated on LB agar plates containing ampicillin (100 μg/l),IPTG (0.5 mM) and X-gal (50 μg/ml). Plasmid DNA was purified frommini-cultures of individual colonies using the BioRobot™ 9600 nucleicacid purification system (Qiagen) and sequenced as described above. TheDNA sequence of the inserts of recombinant clones YCE207, YCE212,YCE216, YCE217, YCE218 and YCE219 could be assembled with the 78 bpgenomic fragment described above into a single contig (see FIG. 2).

Southern blots of the semi-nested 5′ RACE PCR reaction products wereprehybridized at 65° C. for 1 h in modified Church buffer and thenhybridized overnight at 65° C. in the same buffer containing 2×10⁶cpm/ml of the ³²P-labelled insert of clone YCE207. Hybridized blots werewashed at high stringency and autoradiographed. Hybridizing fragmentswere purified from gel, cloned into the pGEM™-T vector (yielding clonesYCE223, YCE224 and YCE226) and sequenced as described above. The DNAsequences of these clones could be assembled with the 78 bp genomicfragment and with clones YCE207, YCE212, YCE216, YCE217, YCE218 andYCE219 into a single contig (FIG. 2). The resulting contig contained anopen reading frame which started at an “ATG” initiation codon andencoded a protein which showed high similarity with the N-terminalsequence of the mouse SEP protein.

To obtain cDNA clones covering the amino-terminal part of the IGS5coding sequence and overlapping with clone YCE59, PCR reactions were setup on human testis Marathon-Ready™ cDNA (Clontech 7414-1) using aspecific forward primer (IP14535; SEQ ID NO:17) based on the 5′ UTRsequence of IGS5 and a specific reverse primer (IP14537; SEQ ID NO:18)located within YCE59. The PCR reaction was performed in a 25 μl volumecontaining 2.5 μl of human testis Marathon-Ready™ cDNA, 2.5 μl ofGeneAmp™ 10×PCR buffer (500 mM KCl, 100 mM Tris pH 8.3, 15 mM MgCl₂,0.01% (w/v) gelatin; PE Biosystems), 1 μl of 10 mM dNTP mix, 10 pmoleseach of the forward and reverse primers and 2.5 units AmpliTaq-Gold™polymerase (PE Biosystems). After an initial denaturation at 95° C. for10 min., PCR reaction tubes were cycled 41× as follows: 1 mindenaturation at 95° C., 1 min annealing at 53° C. and 1 min extension at72° C. PCR reaction products were analysed by agarose gelelectrophoresis. The PCR reaction produced two amplicons of .+-.300 and380 base pairs respectively. The 300 bp and 380 bp fragments werepurified from gel, cloned into the pGEM™-T vector and sequenced asdescribed above. This yielded clones YCE231, YCE233 and YCE235 (300 bpfragment) and YCE229 (380 bp fragment).

Assembly of the DNA sequences of all isolated clones showed the presenceof two types of cDNA sequences, that differed by the presence or absenceof the 78 bp segment, inititially identified within genomic cloneIGS5/S1. These two sequences likely originate from alternatively splicedRNA molecules. The longest transcript contains an open reading frame of2337 nucleotides (encoding a protein of 779 residues) whereas theshorter transcript contains an open reading frame of 2259 nucleotides(encoding a protein of 753 residues). We refer to the coding sequenceand protein sequence of the long form as IGS5DNA1 (shown in SEQ ID NO:3,2340 bp including the stop codon tag) and IGS5PROT1 (SEQ ID NO:4)respectively, whereas the coding sequence and protein sequence of theshorter form are referred to as IGS5DNA2 (shown in SEQ ID NO:5, 2262 bpincluding the stop codon tag) and IGS5PROT2 (SEQ ID NO:6) respectively.Downstream of the postulated methionine initiaton codon within IGS5DNA1and IGS5DNA2 an additional in-frame methionine codon is present at codonposition 10. Although we have opted for the first methionine codon asbeing the initiaton codon some (or even exclusive) initiation oftranslation at codon position 10 cannot be excluded, since bothmethionines appear to be within an equally favorable “Kozak” initiationof translation context (Kozak M., Gene (1999): 234: 187–208). Hydropathyanalysis (Kyte J. et al., J. Mol. Biol. (1982) 157: 105–132; Klein P. etal., Biochim. Biophys. Acta (1985) 815: 468–476) of the IGS5PROT1 andIGS5PROT2 sequences showed the presence of a single transmembrane domainbetween residues 22 to 50. This indicates that IGS5PROT1 and IGS5PROT2are type II integral membrane proteins and thus have a membrane topologysimilar to other members of the NEP/ECE protein family.

TABLE 8 Overview of the oligonucleotide primers that were used inExample 3. SEQ ID NO:7 IP11689: 5′-ACACGGCATCGCTCCTTG-3′ SEQ ID NO:8IP11685: 5′-CCCCCTGGACGGTGAA (C or T) GC (A, C, G or T) T (A or T) (C orT) TA-3′ SEQ ID NO:9 IP12190: 5′-AATCCGTTCACGTTCTGTTCGTCTGCC-3′ SEQ IDNO:10 IP12433: 5′-CCTGGAGGAGCTG (A, C or G) (A, C or T) (A, C, G or T)TGGATG (A or G) A-3′ SEQ ID NO:11 AP1: 5′-CCATCCTAATACGACTCACTATAGGGC-3′SEQ ID NO:12 IP12189: 5′-GTCCTTGCCACCCTCTGCCATCC-3′ SEQ ID NO:13IP12585: 5′-ACCACCCCCGCCCCGATGATCCAGAG-3′ SEQ ID NO:14 IP14241:5′-ACAGCCGGCTAGCAAGGCGTGGCAGCTG-3′ SEQ ID NO:15 IP14242:5′-ACGACAGCCGGCTAGCAAGGCGTGGCAG-3′ SEQ ID NO:16 IP13798: 5′-GG (A, C, Gor T) CT (C or G) ATGGT (A, C, G or T) CT (C or G) CT (C or G) CT (C orG) CT (C or G)-3′ SEQ ID NO:17 IP14535: 5′-CTCCTGAGTGAGCAAAGGTTCC-3′ SEQID NO:18 IP14537: 5′-GCAAACTGGTAGAAGTCGTCACAC-3′

EXAMPLE 4

Alignment of IGS5 with Protein Sequences of Members of the NEP/ECEMetalloprotease Family

For the IGS5 sequence cloned in example 3, homology searches of up todate protein databanks and translated DNA databanks were executed usingthe BLAST algorithm (Altschul S. F. et al, Nucleic Acids Res. (1997)25:3389–3402). These searches showed that IGS5PROT1 was most similar(76% identities over 778 aligned residues) to mouse SEP (GenBankaccession no. AF157105) and also showed 54–55% identities over 696aligned residues to mouse, rat and human neutral endopeptidases(SW:NEP_MOUSE, accession no. Q61391; SW:NEP_RAT, accession no. P07861;SW:NEP_HUMAN, accession no. P08473). Homology searches of IGS5PROT2showed that this sequence was most similar (78% identities over 752aligned residues) to mouse SEP.sup.o (GenBank accession no AF157106). Inanalogy with the mouse SEP and SEP.sup.o proteins it is to be expectedthat IGS5PROT1 and IGS5PROT2 represent the membrane-bound and solubleforms of the IGS5 protein respectively. This is corroborated by thepresence of dibasic residues (KRK) encoded at the 3′ end of the thealternatively spliced 78 bp exon.

Thus, this alignment of the complete IGS5 protein sequence with theother members of the NEP/ECE family shows the relation of IGS5 toNEP/ECE metalloproteases in general, and in particular to the SEP andNEP family members. From this structural alignment it is concluded thatthe IGS5 protein has the functionality of metalloproteases, which inturn are of interest in the context of several dysfunctions, disordersor diseases in animals and humans.

EXAMPLE 5

RNA Expression Analysis of IGS5

IGS5 expression analysis on Human RNA Master Blot™. A solution ofExpress-Hyb™ (Clontech #8015–1) and sheared salmon testis DNA wasprepared as follows: 15 ml of Express-Hyb was preheated at 50–60° C. 1.5mg of sheared salmon testis DNA was heated at 95° C. for 5 minutes andthen quickly chilled on ice. The heat-denatured sheared salmon testisDNA was mixed with the preheated Express-Hyb™. The human RNA MasterBlot™ (Clontech #7770-1) was prehybridised in 10 ml of the solutionprepared above for 30 minutes with contiguous agitation at 65° C. The³²P labelled YCE15 probe (labelled with Prime-it II™ kit, Stratagene)was heat-denatured and added to the remaining 5 ml of the Express-Hyb™solution. Hybridisation was done overnight at 65° C. Washings were donein 2×SSC/1% SDS for 100 minutes (5×20 min.) at 65° C. Two additional 20minutes washes were performed in 200 ml 0.1×SSC/0.5% SDS at 55° C.Finally the Master Blot was autoradiographed using X-ray film.Hybridization of the IGS5 probe on the Master Blot™ showed expression ina wide range of tissues, and in particular expression in testis, smallintestine, prostate and stomach (FIG. 3).

IGS5 Expression Analysis on Human Brain Multiple Tissue Northern BlotsII and IV (#7755-1 and #7769-1 respectively).

An Express-Hyb™ solution (Clontech #8015–1) was preheated at 68° C. Theblot was prehybridised at 68° C. for 1 hour. 100 μg sheared salmontestis DNA was added to the ³²P labelled YCE15 probe (labelled withPrime-it II™ kit, Stratagene) and heat-denatured at 95° C. for 10minutes. The probe was added to the remaining 5 ml of the Express-Hyb™solution and hybridisation was done for 2 hours at 68° C. Washings weredone in 2×SSC/0.05% SDS for 40 minutes (2×20 min.) at RT. Two additional20 minutes washes were performed in 200 ml 0.1×SSC/0.1% SDS at 55° C.The blot was autoradiographed using X-ray film. This Northern blotanalysis showed a major hybridizing band of .+−.3 kb and a minor band of5.5–6 kb in all tissues investigated.

EXAMPLE 6

Expression and Purification of the His-Tagged Ectodomain of Human IGS5

The aim of the experiment was to produce soluble IGS5 protein using thebaculoviral expression system. A recombinant baculovirus was constructedthat expressed the His₆-tagged IGS5 ectodomain upon infection of the Sf9cell-line. Soluble IGS5 protein was then purified from the culturesupernatant in a two step procedure involving lentil-lectin and Zn-IMACchromatography.

We fused the signal peptide of the pro-opiomelanocortin precursor (POMC)to the His-tagged extracellular part of the IGS5 coding sequence. As theenzymatically active site (metalloprotease) of the protein is located atthe C-terminal end, we preferred to add the His-purification tag at theN-terminus of the protein. Furthermore a Gly-Ser linker was insertedbetween the POMC signal peptide and the IGS5 ectodomain. The expressedIGS5 protein started at residue 60 of IGS5PROT2 (SEVC . . . ) and thuscomprised almost the complete IGS5 ectodomain. The cloning strategyinvolved a combination of synthetic oligonucleotide assembly, overlapPCR and 3-points-ligation. This resulted in the expression of a proteinconsisting of the POMC signal (cleaved upon secretion), a Gly-Serlinker, a His6 peptide and the IGS5 extracellular domain.

Example 6a

Construction of the pAcSG2SOLhuIGS5His6 Baculo Transfer Vector

For the construction of the pAcSG2SOLhuIGS5 baculo transfer vector thefollowing DNA fragments were generated:

-   1. The pAcSG2 vector (BD PharMingen) was StuI/NotI digested. The    5527 bp fragment was extracted from agarose gel using the QiaExII    extraction kit (Westburg) and dissolved in 30 μl 10 mM Tris-HCl    pH8.5.-   2. PGEMT clone YCE174 was assembled from clones YCE15, YCE22, YCE64    and YCE65 via a combination of PCR and restriction    digestion/ligation. Primer IP13541, which in contrast to IP11689 did    contain the last 4 nucleotides of the IGS5 coding sequence and the    stop codon, was used in this procedure (Table 9). YCE174 therefore    contained almost the complete coding region of the huIGS5    extracellular domain down to (and including) the stop codon (FIG.    2). YCE174 was XhoI/NotI digested resulting in a 3025 bp, a 1723 bp    and a 448 bp fragment as shown by agarose gel electrophoresis. The    1723 bp fragment, containing the coding region for the huIGS5    ectodomain, was extracted from gel (QiaexII, Qiagen) and dissolved    in 20 μl 10 mM Tris-HCl pH8.5.-   3. A synthetic nucleic acid fragment (180 bp) containing a StuI    recognition site at the 5′ end, followed by the POMC signal    sequence, a Gly-Ser linker, a His6 tag and 65 bp of the 5′ end of    the IGS5 ectodomain coding sequence was assembled by combining the    oligonucleotides IP14165, IP14114, IP14115, IP14116, IP14117,    IP14118, IP14119, and IP14120, followed by overlap PCR with primers    IP14166 and IP14110 (Table 9; see also FIG. 4). The StuI site    present in the natural POMC signal peptide coding sequence was    removed by introducing a silent mutation (IP14115, nucleotide 30    G.fwdarw.A) at bp position 57.

TABLE 9 Overview of the oligonucleotide primers that were used inExample 6. SEQ ID NO:19 IP14165: 5′-GACAAGGCCTATTATGCCGAGATCGTGCTGCAGCCGCTCG-3′ SEQ ID NO:20 IP14114: 5′-AAGGCCAGCAACAGGGCCCCCGAGCGGCTGCAGCACGATC-3′ SEQ ID NO:21 IP14115: 5′-GGGGCCCTGTTGCTGGCCTTGCTGCTTCAAGCCTCCATGG-3′ SEQ ID NO:22 IP14116: 5′-GTGAGAACCGCCACGCACTTCCATGGAGGCTTGAAGCAGC-3′ SEQ ID NO:23 IP14117: 5′-AAGTGCGTGGCGGTTCTCACCATCACCACCATCACAGCGA-3′ SEQ ID NO:24 IP14115: 5′-AGCCAGGGGTGGTGCAGACCTCGCTGTGATGGTGGTGATG-3′ SEQ ID NO:25 IP14119: 5′-GGTCTGCACCACCCCTGGCTGCGTGATAGCAGCTGCCAGG-3′ SEQ ID NO:26 IP14120: 5′-GGGTCCATGTTCTGGAGGATCCTGGCAGCTGCTATCACGC-3′ SEQ ID NO:27 IP14166: 5′-GACAAGGCCTATTATG-3′ SEQ IDNO:28 IP14110: 5′-GGGTCCATGTTCTG-3′ SEQ ID NO:29 IP14111:5′-AGCGAGGTCTGCAC-3′ SEQ ID NO:30 IP14112: 5′-GTAGATGATGTGCCG-3′ SEQ IDNO:31 IP13541: 5′-GCACTAGTCTTGGCTACCACACGCG GCATCGCTCCTTG-3′Assay buffer: 100 mM Tris pH 7.0, 250 mM NaCl. All test compounds weredissolved in DMSO at 10 mM and were further diluted with assay buffer.

Example 7b

Assay Procedure

A quantity of 70 μl of the assay buffer, of 10 μl enzyme workingsolution and of 10 μl test compound solution were mixed in an Eppendorfvial and preincubated at 37° C. for 15 minutes. Then, 10 μl substratestock solution was added and the reaction mixture was incubated at 37°C. for 60 minutes to allow for enzymatic hydrolysis. Subsequently theenzymatic reaction was terminated by heating at 95° C. for 5 minutes.After centrifugation (Heraeus Biofuge B, 3 min) the supernatant wassubjected to HPLC analysis.

Example 7c

HPLC Procedure

In order to separate the remaining substrate from the cleavage productsreversed phase HPLC technique was used with a CC 125/4 Nucleosil 300/5C₁₈ RP column and a CC 8/4 Nucleosil 100/5 C₁₈ precolumn (commerciallyavailable from Macherey-Nagel, Duren, Germany). Thus, 60 μl of thereaction samples obtained in Example 7b were injected into the HPLC, andthe column was eluted at a flow rate of 1 ml/min by applying thefollowing gradient and solutions:

Solution A: 100% H₂O + 0.5 M H₃PO₄, pH = 2.0 Solution B: 100%acetonitrile + 0.5 M H₃PO₄  0–2 min 20% B  2–6 min 20–60% B  6–8 min 60%B  8–10 min 60–90% B 10–13 min 90% B 13–15 min  90–100% BPeptides were detected by absorbance at 214 nm and by fluorescence withan excitation wavelength of 328 nm and an emission wavelength of 393 nm.

Example 7d

Calculations

The increasing fluorescence signal of the HPLC-peak of the peptide withthe unquenched Mca-fluorophor after hydrolysis was taken as the basisfor any calculation. This signal was compared for the samples with andwithout inhibitor and % inhibition was calculated on basis of therespective peak areas.% inhib=100*(1−A _(inhib) /A _(control))All samples were run in duplicate and mean values were used. A standardinhibitor (10 nM and 100 nM Phosphoramidon) and a solvent control (0.1%)was added to each assay run.

Example 7e

Results

With regard to the IGS5 polypeptides of the present invention theresults of Example 7 show that these IGS5 metalloprotease polypeptideshydrolyze in vitro a variety of vasoactive peptides known in the stateof the art, in particular such as Big ET-1, ET-1, ANP and bradykinin.The results of the hydrolysis assay in comparison to the activity of SEPare shown in Table 10. From these results it is concluded that IGS5 maybe particularly involved in the metabolism of said biologically activepeptides.

TABLE 10 Hydrolysis of vasoactive peptides by IGS5 polypeptides incomparison to SEP (soluble secreted endopeptidase). % Hydrolysis %Hydrolysis by IGS5 Polypeptides by SEP (Emoto et al.) Conditions:Conditions: 100 μg IGS5 polypeptide; 10 μg SEP; Vascoactive 0.5 μMsubstrate; 2 h, 0.5 μM substrate; 12 h, Peptide 37° C. 37° C. ANP  5(80*) >95 Bradykinin 100 (62**) >95 Big ET-1 (?)*** 42 ET-1 30 92Substance P n.d. >95 Angiotensin 1 n.d. >95 17 aa Big ET 41 n.d. *500 μgIGS5 polypeptide **10 μg IGS5 polypeptide ***activity was detected butcould not be quantified due to problems with the HPLC-detection

Furthermore, the results of the experiments with reference compounds forinhibition of ECE- and/or NEP-activity show that the activity of IGS5metalloprotease polypeptides of the present invention to convert theBig-ET-1 analogue 17 aa Big-ET is inhibited by phosphoramidon, areference compound for ECE/NEP-inhibition, but IGS5 is not efficientlyinhibited by the NEP-inhibitor thiorphan. These results are shown inTable 11. IGS5 polypeptides are also not inhibited by the selectiveECE-inhibitor CGS-35066, a potent and selective non-peptidic inhibitorof endothelin-converting enzyme-1 with sustained duration of action. (DeLombart et al., J. Med. Chem. 2000, Feb. 10; 43(3):488–504).

TABLE 11 Inhibition of IGS5 polypeptide's activity to convert theBig-ET-1 analogue 17 aa Big-ET. Inhibitor Compound IC₅₀ nMPhosphoramidon 18 Thiorphan >1000 CGS-35066 1300

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the described embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

1. A method for screening for a compound which influences the activityof a polypeptide having a zinc metalloprotease activity and consistingof an amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4 and SEQ ID NO:6, the method comprising: (a) contactinga preparation comprising the polypeptide with a candidate compound inthe presence of a substrate for the polypeptide; and (b) assessingwhether the candidate compound results in a stimulation or inhibition ofthe activity of the polypeptide.
 2. A method according to claim 1,wherein the preparation comprises a cell expressing the polypeptide. 3.A method according to claim 1, wherein the candidate compound is mixedwith a solution containing the polypeptide and a suitable substrate toform a mixture, an activity of the polypeptide in the mixture ismeasured and the measured activity is compared with the activity of thepolypeptide in a control solution without the candidate compound.